Compositions and vaccines comprising vesicles and methods of using the same

ABSTRACT

The disclosure relates to compositions, pharmaceutical compositions, vaccines, and methods of making and using the same. The compositions of the disclosure are useful to stimulate an antigen-specific immune response and, in some embodiments, a protective immune response in an animal after challenge to pathogens. The compositions of the disclosure are also useful for treatment and prevention of disease in a subject such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser. Nos. 61/830,435, filed Jun. 3, 2013, and 61/834,311, filed Jun. 12, 2013, and 61/916,564 filed Dec. 16, 2013, and 61/972,992 filed Mar. 31, 2014 each of which are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

The invention was made with government support under CTS0608906 awarded by National Science Foundation and 5R01AI68888 & AI057168 awarded by the National Institutes of Health. The government may have certain rights in and to this invention.

TECHNOLOGY FIELD

The present invention relates generally to catanionic vesicles created by applying cationic and anionic surfactants to pathogenic bacteria, resulting in vesicles which display molecular components of the bacteria on their exterior surface. More specifically, the inventions relates to compositions and methods for the creation and administration of catanionic vesicles displaying molecular components of bacterial antigens for use as a vaccine.

BACKGROUND

In many types of bacteria, cell to cell recognition is a key feature of pathogen virulence. Accordingly, methods for the extraction and purification of carbohydrates and proteins from cellular membranes, followed by reconstitution of cellular components into stable hydrophobic matrices has been widely used. Due to the complexity of biological membranes, cell surface components are often arranged in artificial membranes such as liposomes.

Outer membrane proteins from bacteria have been incorporated into liposomes for vaccines and the immune response of the resulting liposome preparations is known in the art. Liposomal formulations of antigens have significant advantages in vaccine formulation over cell-based formulations since they avoid safety concerns that may arise from attenuated or killed pathogens. However, methods of liposomal formulations of cell surface components still have limitations as vehicles for displaying cell surface proteins and lipids in a stable membrane-like environment. First, since cell surface components typically reside in the hydrophobic bilayer, the solubilization and purification of these substances from the membrane often results in denaturation of the protein. Second, it is unclear that insertion of the purified protein into the membrane of a liposome results in the presentation of the protein in its ‘natural’ form, especially in the case of denatured components.

Finally, liposomal formulations are limited by the general physicochemical characteristics of the liposomes themselves. The production of liposomes requires either sonication or passage through a membrane, and these processes result in the addition of mechanical stress to the system that also may lead to denaturation of sensitive biological components. Liposomal formulations prepared by either sonication or membrane extrusion are very heterogeneous in size, with distributions ranging from 300 nm to 20 microns. Finally, liposomal formulations are difficult to maintain in long term storage because they often precipitate within days of creation due to their inherent thermodynamic instability, typically compounded by additional instability in biological media due to pH and ionic strength issues.

In addition to liposomal preparations, current methods of making multivalent vaccines against Neisseria involve removal of lipooligo and lipopolysaccharides (LOS and LPS respectively) specifically because of their toxicity and immunological problems. van der Waterbeemd et al., Vaccine 28 (2010) 4810-4816 discloses that outer membrane vesicles used as vaccines maintain a residual amount of LPS (only about 1%) but are needed to adjuvate the immune response. The removal of LPS, however, also depletes the amount of lipoprotein that exists in the vesicles and reduces immunogenicity.

In contrast to van der Waterbeemd et al., the present disclosure addesses the limitations of vaccine and composition delivery via liposomes by using artificial membrane components from catanionic surfactants that package pathogen antigens and antigens associated with hyperproliferative disease, such as cancer. The vesicles also protect membrane-bound antigens from degradation as well as cloak or reduce the toxicity caused by bacterial lipopolysaccharides and lipooligosaccharides.

Francisella tularensis is an immune-evasive coccobacillus that causes tularemia disease in humans and animals. Francisella tularensis is classified as a Tier 1 agent. To date, there is no vaccine for Francisella tularensis approved by the FDA. Limitations in Francisella tularensis vaccine development include the risk of reversion of live mutant strains and poor immunogenicity of killed bacteria. The present disclosure provides a multivalent vaccine from whole cell extract that can be used for treatment and/or prevention of bacterial infection while effectively adjuvating the immune response.

SUMMARY OF THE INVENTION

The disclosure relates to catanionic vesicles comprising LPS or LOS in therapeutically effective amounts. The disclosure also relates to catanionic vesicles and vaccines comprising catanionic vesicles that comprise Francisella tularenis components and/or antigens. In some embodiments, the disclosure relates to a methods of treating and/or preventing tularemia in a subject in need thereof by administering any of the disclosed vesicles, pharmaceutical compositions, or vaccines in a therapeutically effective amount to reduce or eliminate symptoms of tularemia or bacterial infection. The disclosure also relates to catanionic vesicles and vaccines comprising catanionic vesicles that comprise Neisseria gonorrhroeae components and/or antigens. In some embodiments, the disclosure relates to a methods of treating and/or preventing bacterial infection in a subject in need thereof by administering any of the disclosed vesicles, pharmaceutical compositions, or vaccines in a therapeutically effective amount to reduce or eliminate symptoms of the bacterial infection or bacterial infection in the subject.

In some embodiments, the disclosure relates to a methods of treating and/or preventing malignant cell growth in a subject in need thereof by administering any of the disclosed vesicles, pharmaceutical compositions, or vaccines in a therapeutically effective amount to reduce or eliminate symptoms of cancer or growth of malignant cells in the subject.

The present disclosure relates to a composition comprising a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins. In some embodiments, the at least one or plurality of bacterial immunogens is from a Gram-negative bacterial cell. In some embodiments, the at least one or plurality of bacterial immunogens is derived from cells that are from Neisseria or Franicsiella. In some embodiments, the at least one bacterial immunogen is from whole bacterial cell extract. In some embodiments, the one or plurality of bacterial immunogens comprise one or a combination of pilin, porA, porB, OPA, DnaK, Tul4, and FopA. In some embodiments, the one or plurality of bacterial immunogens comprises Pan HLA-DR reactive epitope (PADRE) individually or conjugated to one or a plurality of membrane-bound sugar molecules. In some embodiments, the catanionic surfactant vesicle is lyophilized or frozen.

The disclosure relates to a pharmaceutical composition comprising: a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins and/outermembrance bacterial proteins; and a pharmaceutically acceptable carrier or excipient.

The disclosure relates to a vaccine or a pharmaceutical composition comprising: a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins; and a pharmaceutically acceptable carrier or excipient.

The disclosure relates to vaccine or a pharmaceutical composition comprising: a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins; a pharmaceutically acceptable carrier; and a nucleic acid molecule encoding one or a plurality of immunogens and or immunomodulating proteins, a protein adjuvant, a synthetic adjuvant, an attenuated bacterial cell, or a recombinant bacterial cell comprising a nucleic acid molecule encoding one or a plurality of immunogens and or immunomodulating proteins.

The disclosure relates to a method of treating and/or preventing a pathogen infection in a subject in need thereof, comprising: administering to the subject the pharmaceutical composition comprising: a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of pathogen immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins or bacterial membrane-bound proteins; and a pharmaceutically acceptable carrier.

The disclosure relates to a method of immunizing a subject comprising: administering to a subject in need thereof a therapeutically effective amount of the vaccine or a pharmaceutical composition comprising: a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins; and a pharmaceutically acceptable carrier. In some embodiments, the effective amount is an amount effective to elicit an immune response. In some embodiments, the immune response is a protective, antigen-specific immune response after challenge of the subject with a pathogen.

The disclosure relates to a method of integrating a bacterial antigen into a vesicle, comprising:

-   -   (a) centrifuging bacterial cells;     -   (b) removing any bacterial growth medium;     -   (c) adding a weight/weight molar ratio of cationic surfactant         and anionic surfactant in aqueous solution to the bacterial         cells;     -   (d) stirring resulting mixture at room temperature for at least         about 30 minutes;

wherein the method does not comprise a step of purification prior to steps (a)-(d).

In some embodiments, the cationic surfactant and anionic surfactant are SDBS and CTAT, respectively, at a molar weight ratio of about 70:30 or about 30:70. In some embodiments, the vesicle, composition or vaccine comprises at least one or a plurality of bacterial antigens from Neisseria and/or Francisella. In some embodiments, methods comprise encapsulating at least one or a plurality of bacterial antigens from Neisseria and/or Francisella. In some embodiments, the aqueous solution or compositions comprise a marker or active agent. In some embodiments, the method further comprises filtering the vesicles through a size exclusion or ion chromatography column.

The disclosure relates to a method of creating a library of catanionic vesicles comprising:

-   (a) adding a weight/weight molar ratio of cationic surfactant and     anionic surfactant in aqueous solution to a population of bacterial     cells in pelleted form; -   (b) stirring resulting mixture at room temperature for at least     about 30 minutes;

wherein the method does not comprise a step of purification prior to steps (a)-(b). In some embodiments, the cationic surfactant and anionic surfactant are SDBS and CTAT, respectively, at a molar weight ratio of about 70:30. In some embodiments, the bacterial cells comprise at least one or a plurality of bacterial antigens from Neisseria and/or Francisella. In some embodiments, the aqueous solution comprises one or more of a dye, a fluorescent protein, or a chemiluminescent agent.

The disclosure relates to a method of integrating a bacterial antigen into a vesicle, comprising:

-   -   (a) centrifuging bacterial cells;     -   (b) removing any bacterial growth medium;     -   (c) adding a weight/weight molar ratio of cationic surfactant         and anionic surfactant in aqueous solution to the bacterial         cells;     -   (d) stirring resulting mixture at room temperature for at least         about 30 minutes;

wherein the method does not comprise a step of purification prior to steps (a)-(d), wherein the method further comprises filtering the vesicles through a size exclusion or ion chromatography column.

The disclosure relates to a method of manufacturing an antibody against an antigen comprising injecting into a subject the composition of comprising a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins and/or bacterial cell membrane proteins; and subsequently collecting its serum.

The disclosure relates to a method of reducing the immunological toxicity of a bacterial antigen comprising loading the bacterial antigen into a catanionic vesicle of comprising a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises at least about 15 micrograms per microliter of bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) optionally comprising one or a plurality of bacterial lipoproteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Total protein and carbohydrate concentrations in vesicle extract samples determined by colorimetric BCA and carbohydrate assays. All five extraction methods yielded similar quantities of protein and carbohydrate by methods (1) solid surfactants added to the bacterial cell pellet followed by the addition of water, (2) SDBS solution added to the bacterial cell pellet followed by the addition of a CTAT solution (3) CTAT solution added to bacterial cell pellet followed by the addition of solid SDBS (4) SDBS solution added to bacterial cell pellet followed by the addition of solid CTAT (5) bare vesicles added to the bacterial cell pellet. BCA assay indicating the presence of protein in the different extraction methods.

FIG. 2: Vesicle-containing fractions purified by gel filtration followed by silver staining. Lanes 1-6 and 9 were loaded with 1 μL, Lane 7 was loaded with 5 μL, and Lane 8 was loaded with 167 nL of sample and analyzed by SDS-polyacrylamide Tris-tricine 16.5% v/v gels followed by silver staining. Lanes 1-6 correspond to vesicle-containing fractions from prepared from extraction methods 1-6, respectively, and show similar protein patterns. Lane 7 shows the purified cell lysate disrupted by SDBS and purified by gel filtration. Lane 8 contains a very different protein pattern from resuspended GC cell pellet. Lane 9 contains purified LOS F62ΔlgtD as a standard.

FIG. 3: Vesicle extract fractions from gel filtration analyzed by silver staining. Lanes were loaded with 1 μL of sample and analyzed by SDS-polyacrylamide Tris-tricine 16.5% v/v gels followed by silver staining. Lanes 1 and 2 represent the void volume and are free of protein and carbohydrate. Lanes 3 and 4 correspond to vesicle containing fractions and indicate the presence of a range of proteins and a high concentration of the carbohydrate LOS F62ΔlgtD with the darkest band at the bottom of the gel. Lanes 5-14 show the diminishing presence of proteins and LOS.

FIG. 4: Proteomics data from GC/MS of vesicle extract samples.

FIG. 5: Western blotting of vesicle-containing fractions (lane 2), lysate, and supernatant. The purified vesicle extract shows antibody binding to several proteins and LOS, specifically porin (36 kD) and OPA (25-30 kD). The lysate fraction does not show any antibody binding, indication that vesicles contain surface antigens.

FIG. 6: Proteinase K digestion. Lanes 1 and 3 show the protein pattern of the GC lysate and vesicle extract, respectively, after silver staining. Lanes 2 and 4 contain GC lysate and vesicle extract purified by gel filtration and show digestion of all protein bands and retention of LOS at the bottom of the gel.

FIG. 7: Protection from trypsin digestion. GC lysate and surfactant GC vesicle extract samples.

FIG. 8: Chemical structure of lipooligosaccharide components from the Gramnegative bacteria N gonorrhoeae. The various regions include the 0-antigen, core, and lipid A.

FIG. 9: Immune response of LOS vs. C12-PADRE/LOS conjugated catanionic vesicles. Vesicles loaded with only carbohydrate would generate IgM antibodies. Addition of the epitope C12-PADRE to LOS functionalized vesicles would generate IgG antibodies.

FIG. 10: Preparation of LOS conjugate from N gonorrhoeae. The toxic lipid A portion was cleaved from the oligosaccharide using hot acetic acid and a hydrophobic C12-linker was added.

FIG. 11: Chemical structure of lipooligosaccharide (LOS) F62˜lgtD purified from N gonorrhoeae. 0-Antigen: Gal˜1-4GlcNAc˜1-3Gal˜1-; Core: −4Glc˜1-4Hepal-(−3Hepal-GlcNAcal)5Kdo4-(−2aKdo); lipid A.

FIG. 12: Chemical (AKFVAAWTLKAAA SEQ ID NO:1). structure of unconjugated PADRE peptide.

FIG. 13: Synthesis of C12-PADRE peptide conjugate. Conjugated PADRE peptide contains a hydrophobic moiety, which allows for incorporation into the membrane of vesicle bilayers.

FIG. 14: Average amount of carbohydrate in TRIAD vaccine from two batches. Carbohydrate determined by phenol/sulfuric acid colorimetric assay.

FIG. 15: Preparation of TRIAD vaccine formulated with catanionic surfactant vesicles containing the C12-PADRE peptide conjugate and LOS from N gonorrhoeae. Ratio of conjugate components can be adjusted during vesicle formation to control the surface decoration of antigens.

FIG. 16: Antibody titer results in mice inoculated with LOS and LOS/C12-PADRE functionalized surfactant vesicles; performed by a student in the Stein group (unpublished results).

FIG. 17: Chemical structure of one type of lipopolysaccharide (LPS) from F. tularensis.

FIG. 18: Synthesis of N-dodecanoylsuccinimide.

FIG. 19: Synthesis of dodecanoic acid tethered to PADRE peptide.

FIG. 20: Functionalized catanionic vesicles displaying F. tularensis LVS LPS as the only F. tularensis component are protective against F. tularensis LVS challenge, but fail to induce robust IgG antibody responses. (A.) Schematic of experimental protocol. Mice were injected twice, two weeks apart (D −28, D −14), by intraperitoneal injection (i.p.), with either purified F. tularensis LVS LPS in its soluble form (LPS), purified F. tularensis LVS LPS included in surfactant vesicles (LPSvesicles), the T cell epitope, C12-PADRE, covalently coupled to surfactant vesicles (PADRE-vesicles), or purified F. tularensis LVS LPS and C12-PADRE covalently coupled to vesicles (LPS-PADRE-vesicles). Two weeks after the second immunization, all mice were challenged i.p. with about 6000 colony forming units (CFU) F. tularensis LVS per mouse. (B.) Survival of individual mice after the F. tularensis LVS challenge. (C.) Sera were collected prior to each immunization, prior to challenge, and on the 11th day following challenge. Sera from each time point were pooled, except for serum from the last time point in which each survivor was bled individually. Antibody titers to whole F. tularensis LVS were measured by FACS-based bead assay utilizing isotype-specific secondary antibodies for detection as previously described (Cole et al., PNAS 106. 2009.). Error bars represent standard error of the mean for 5 mice. The results are representative of one of two separate experiments with similar outcomes.

FIG. 21: LVS-V nanoparticles fully protect mice against F. tularensis (Ft) LVS challenge. (A to C) In experiment 1, mice were injected twice, 2 weeks apart (days −28 and −14), i.p. with sterile saline (white square, dotted line), catanionic vesicles formed in the presence of F. tularensis LVS (LVS-V, 35 μg protein) (black diamond, solid line), or the equivalent amount of bare catanionic vesicles (V) (black triangle, dashed line). Two weeks after the second immunization, all mice were challenged i.p. with 30,000 CFU F. tularensis LVS per mouse. (D to F) In experiment 2, the challenge dose was increased to 70,000 CFU F. tularensis LVS per mouse. (G to I) In experiment 3, the time after final immunization was increased to 4 weeks. Everything else was kept the same as for experiment 1. In each experiment, the percent starting weight (A, D, and G), and the severity of tularemia symptoms (B, E, and H), from 0 for healthy to 5 for dead, and survival of the individual mice following challenge (C, F, and I) are shown. Clinical scores were assigned as follows: for mice infected with F. tularensis LVS, a score of 0 was assigned for healthy mice with normal behavior (exploring cage, feeding, alert), 1 for mild illness (usually marked by lower activity and weight loss), 2 for mild-moderate illness (mice showing symptoms of score 1 plus piloerection), 3 for moderate-severe illness (mice showing symptoms of score 2 plus assuming a hunched posture), 4 for severe illness (mice showing symptoms of score 3 and minimal activity or blepharitis [crustiness around the eye] involving one or both eyes), 4+ for moribund illness (mice showing symptoms of score 3 and were nonresponsive to stimulation [these mice were euthanized]), and 5 for mice found dead in their cage. Mice with scores of 4+ and 5 were reported on the day of death only and were excluded from analysis on subsequent days. The arithmetic means+/−the standard errors of the means (SEM) of the clinical scores were reported.

FIGS. 22 A-D: LVS-V induce robust antibody responses where IgM is essentially all anti-LPS antibody and IgG is predominantly directed against non-LPS epitopes. (FIG. 22A) Schematic of immunization and bleeding schedule. The mice were immunized and challenged as described in FIG. 21. The mice were challenged again 22 days following the primary challenge to determine the effect on antibody responses. (FIG. 22B) ELISA data measuring F. tularensis-specific IgM. (FIG. 22C) ELISA data measuring F. tularensis specific IgG (all subclasses). Microwell plates were coated with purified F. tularensis LVS LPS (top panels) or whole F. tularensis LVS bacteria (bottom panels) to distinguish between LPS-specific and overall anti-F. tularensis antibody levels. The titers are shown on a log scale. Each symbol represents one mouse. ND, none detected. (FIG. 22D) The geometric means of the ELISA titers detected in B and C were replotted for easier visual comparison between the antibody levels directed against whole F. tularensis LVS (filled symbols, solid lines) and those against purified F. tularensis LVS LPS (open symbols, dashed lines) in both IgG (black square, black) and IgM (black triangle, gray) assays. **, p<0.01; ***, p<0.001.

FIG. 23: Passive immunization protects against F. tularensis LVS challenge. (A) IgM and IgG titers against whole F. tularensis LVS were measured by ELISA in pooled donor serum of saline-immunized mice (control serum) or of mice previously immunized twice i.p. with empty vesicles or LVS-V (x axis). *, p<0.05 by Student t test. (B to D) Sixty microliters of serum was transferred to naive 6- to 8-week-old mice by tail vein injection 1 day prior to challenge with 100,000 CFU F. tularensis LVS. The percent starting weight (B), severity of clinical symptoms (C), and survival (D) were measured following challenge. The combined data from two independent experiments are shown (n=7). The error bars represent the standard errors of the means (SEM). *, p<0.05; ***, p<0.001 by log rank test.

FIG. 24: Comparison of cytokine gene and protein expression induced early after empty vesicle versus LVS-V administration. The mice were injected i.p. with saline, LVS-V (35 μg protein), or the equivalent amount of empty vesicles. (A) Four hours after vaccination, each mouse was euthanized and the liver harvested. Gene induction of iNOS and cytokine and chemokine genes representative of inflammatory responses was measured by qRT-PCR. Each symbol represents an individual mouse. (B) After immunization, mice were bled at the indicated time points and cytokine protein levels were measured in the serum samples of individual mice by the Luminex assay. The average serum concentrations±the SEM of 5 mice are shown for KC and IL-6.

FIG. 25: Surface charge, but not size, of vesicles is affected by the F. tularensis protein content of the nanoparticles. The vesicles prepared from different sized bacterial pellets (as described in the Methods) were analyzed for (A.) zeta potential and (B.) average radius (solid squares). In addition, the same analysis was carried out for multiple individually prepared preparations of catanionic vesicles produced in the presence of F. tularensis Schu S4 strain (Schu S4-V) (gray triangles). Each data point represents a distinct batch of vesicle vaccine. (C.) Silver stain and (D.) Western analysis of four independent batches of Schu S4-V.

FIG. 26: LVS-V are made with a high degree of consistency and are immunogenic. F. tularensis LVS molecules were incorporated into surfactant vesicles to form LVS vesicles. Lysates derived from a sampling of the F. tularensis bacterial pellets prior to the addition of surfactants (Ft LVS) and empty control vesicles (V) were also included for comparison. (A) Samples were separated by SDS PAGE on a 10 to 20% gradient gel and silver stained for total protein. Asterisks and filled circles mark bands that were extracted with low and high efficiency into vesicles, respectively. (B) The same samples were subjected to Western analysis (WB) with sera from mice that had been immunized twice with LVS-V (prechallenge) and challenged twice with F. tularensis LVS (postchallenge) as described in FIG. 22. The silver stain and serum blots are representative of 8 separate experiments.

FIG. 27: Catanionic vesicles associate with bacterial LPS in addition to the proteins, and addition of protease inhibitors does not significantly affect the components incorporated into the vesicles. F. tularensis LVS molecules were incorporated into surfactant vesicles (V) to form LVS-vesicles (LVS-V). Lysates derived from a sampling of the bacterial colonies prior to the addition of surfactants was also included for comparison (Ft LVS). (A.) Samples were separated by SDS-PAGE on a 10-20% gradient gel and were subjected to Western analysis with rabbit antibodies directed against F. tularensis LVS LPS. (B.-C.) V, LVS-V, and F. tularensis LVS lysates were produced in the absence or presence of Roche Complete protease inhibitor cocktail. Samples were separated by SDS-PAGE on a 4-20% gradient gel and compared by (B.) Western analysis with mouse sera of LVS-V-immunized mice harvested two weeks after the second immunization, prior to F. tularensis LVS challenge. (C.) Samples were also compared by silver staining.

FIG. 28: Serum samples from mice immunized with LVS-V or Schu S4-V and challenged with live homologous bacteria detect similar bands by Western analysis but also exhibit differences in their binding specificities. (A) Mice were immunized either with LVS-V by two i.p. injections and challenged i.p. with F. tularensis LVS as described in FIG. 21 or immunized with Schu S4-V by i.p. and i.n. routes and challenged i.n. with F. tularensis Schu S4. Serum samples harvested 3 weeks postchallenge (LVS serum, 1:10⁶; Schu S4 serum, 1:10⁵) were used to probe Western blots (4 to 20% gradient gel) of vesicles (V, LVS-V, and Schu S4-V), whole-lysed bacteria (LVS and Schu S4), and purified recombinant F. tularensis proteins (DnaK and Tul4). (B) The same membrane at a lower (1−s) exposure to show DnaK and Tul4 detected by Schu S4-V-immunized/Schu S4-challenged survivor serum. Similar results were seen in two separate experiments.

FIG. 29: Prominent F. tularensis epitopes detected in serum samples from vesicle-immunized mice are conserved across multiple strains of F. tularensis. F. tularensis from strains Schu S4, MA00-2987 (A1 strains), WY96-3418 (A2 strain), KY99-3387, OR96-0246, and LVS (B strains) were grown in enriched TSB and subjected to Western analysis (4 to 20% gradient gel) with the antisera used in FIGS. 26 and 28. (A) Sera from LVS-V-immunized mice before challenge. (B) Sera from LVS-V-immunized mice after live LVS challenge. (C) Sera from Schu S4-immunized mice after live Schu S4 challenge.

FIG. 30: Outcome of immunized mice after live intranasal F. tularensis Schu S4 challenge. a Mice were immunized with control vesicles (V) or Schu S4-V at 2-week intervals by the indicated routes. Two weeks after the final immunization, all mice were challenged with −20 CFU (ranging from 3 to 49 CFU in different experiments) of F. tularensis Schu S4 by the intranasal route. b i.p., intraperitoneal; i.n., intranasal; s.c., subcutaneous. c Mean and standard deviation of time to death were calculated from only those animals that succumbed to infection. d Fisher's exact test, P value compared to that of control; NS, not significant. e One-way ANOVA, p value of Tukey posttest compared to that of control.

FIG. 31: Mice immunized with Schu S4-V show a delay in clinical symptoms after i.n. challenge with Ft Schu S4 and the route of immunization affects IgG titer. (A.) Clinical scoring of individual mice on indicated days: Green (0)—healthy, Yellow (1)—mild illness, Light Orange (2)—moderate illness, Dark orange (3)—severe illness, Red (4)—moribund or dead, Black Hatch (5)—cumulative total of dead mice in each group. These results represent a single representative experiment in which all mice were assessed twice daily. (B.) Ft specific IgG titers from pooled sera obtained one week after the second and third immunizations (mean and SEM).

FIG. 32: Targeting of catanionic surfactant vesicles to cells.

FIG. 33: Retention of doxorubicin over fifteen days in catanionic surfactant vesicles. Concentration of doxorubicin vesicle stock solution was 33 μM. After purification by SEC, vesicle-containing fractions contained ˜23 μM of the drug.

FIG. 34: Table 5.2. Amount of doxorubicin in vesicles from increasing concentrations.

FIG. 35: WST −1 cell proliferation assay on HepG2 cells treated with bare vesicles and doxorubicin loaded vesicles. Cells were treated for 72 h. Bare vesicles IC50=58 μg/mL and doxorubicin vesicles IC50=51 μg/mL.

FIG. 36: WST −1 cell proliferation assay on HepG2 cells treated with doxorubicin. Free drug was incubated with cells for 72 h. IC50=0.16 μg/mL.

FIG. 37: Free doxorubicin compared to doxorubicin loaded catanionic vesicles incubated with normal cells.

FIG. 38: Doxorubicin loaded untargeted and targeted catanionic vesicles incubated with normal cells vs. cells that over express a receptor.

FIG. 39: Chemical structure of C12-folate conjugate.

FIG. 40: WST −1 cell proliferation assay on A549 cells treated with folate targeted catanionic vesicles. Cells were treated for 72 h. Folate vesicles IC50=7.2 μg/mL, Folate-DOX vesicles IC50=5.6 μg/mL.

FIG. 41: WST −1 cell proliferation assay on ovarian IGROV-1 cells, which over express folate, treated with folate targeted catanionic vesicles. Cells were treated for 72 h. Folate vesicles IC50=3.4 μg/mL, Folate-DOX vesicles IC50=2.2 μg/mL.

FIG. 42: FIG. 5.11. Fluorescently-labeled vesicles binding with cells.

FIG. 43: Chemical structure of maytansine.

FIG. 44: Absorbance of a maytansine analogue and maytansine loaded vesicle fraction from SEC. The maytansine analogue absorbs light at 290 nm.

FIG. 45: Chemical structure of paclitaxel fluorescein derivative.

FIG. 46: Absorbance of paclitaxel loaded vesicles at 445 nm. After purification by SEC, vesicle-containing fractions contained 0.9 μM of drug.

FIG. 47: Table of protein content and sugar content of vesicles after using two disclosed techniques of vesicle encapsulation of bacterial antigens. In Method 4-I, 30 mg of CTAT, 71 mg SDBS, 6.9 mL of Millipore water, and 3 mL of cell suspension were mixed and then stirred for 15 minutes. The mixture was centrifuged for 1 hour and the supernatant was decanted. 1 mL of the sample of supernatant was purified on the column. In Method 4-II, 9.9 mL of preformed vesicles (1% SDBS rich), 3 mL cell suspension, and 5.91 mL of Millipore water were mixed and then stirred for 1 hour. The mixture was centrifuged for 1 hour and the supernatant was decanted. 1 mL of the sample of supernatant was purified on the column.

FIG. 48: The proteins preferentially extracted using the various methods were further analyzed by gel electrophoresis. Silver staining of vesicles derived from extraction of Neisseria gonorrhoeae by Methods 4-I and 4-II are provided.

FIG. 49. Mice were immunized twice, i.p./i.p., with either Schu S4-V or LVS-V. Western blots (4-20% gradient gel) of bacterial lysates from well-characterized Ft LVS deletion mutants and WT Ft strains were probed with sera harvested 2 weeks after immunization. Mice were not challenged with live bacteria.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.

The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the term “associated with” refers to the state of two or more entities which are linked by a direct or indirect covalent or non-covalent interaction. In some embodiments, an association is covalent. In some embodiments, a covalent association is mediated by a linker moiety. In some embodiments, an association is non-covalent (e.g. charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.). For example, in some embodiments, an entity (e.g. targeting moiety or therapeutic agent to be delivered) may be covalently associated with a particle. In some embodiments, an entity (e.g. targeting moiety or therapeutic agent to be delivered) may be non-covalently associated with a particle, (e.g. the entity may be associated with the surface of, encapsulated within, surrounded by, and/or distributed throughout a vesicle or particle disclosed herein).

The term “anhydrous” is used throughout the specification to describe a form in which the purgative salts according to the present invention can be administered. Anhydrous formulations are those which essentially have excluded water from the formulations, except, in such instances where the salt is hydrated or otherwise complexed with small amount of water.

The term “salt” refers to acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Salts according to the present invention may be used in a variety of forms, for examples anhydrous or a hydrated crystalline form. In some embodiments, the salts may be those that are physiologically tolerated by a patient.

The term “soluble” or “water soluble” refers to an aqueous solubility that is higher than 1/10,000 (mg/mL). The solubility of a substance, or solute, is the maximum mass of that substances that can be dissolved completely in a specified mass of the solvent, such as water. “Practically insoluble” or “insoluble,” on the other hand, refers to an aqueous solubility that is 1/10,000 (mg/mL) or less.

The terms “treating” and “to treat”, mean to alleviate symptoms, eliminate the causation either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms. The term “treatment” includes alleviation, elimination of causation (temporary or permanent) of, or prevention of symptoms and disorders associated with any condition. The treatment may be a pre-treatment as well as a treatment at the onset of symptoms.

“Effective amount” refers to an amount of a compound, material, or composition, as described herein effective to achieve a particular biological result such as, but not limited to, biological results disclosed, described, or exemplified herein. Such results may include, but are not limited to, the effective reduction of symptoms associated with any of the disease states mentioned herein, as determined by any means suitable in the art. The effective amount of the composition may be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, the type and/or severity of the particular condition being treated, or the need to modulate the activity of the molecular pathway induced by association of the analog to its receptor. The appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art.

Sequence homology for nucleotides and amino acids may be determined using PASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP* 4.0b1 0 software (D. L. Swofford, Sinauer Associates, Massachusetts). “Percentage of similarity” is calculated using PAUP* 4.0b1 0 software (D. L. Swofford, Sinauer Associates, Massachusetts). The average similarity of the consensus sequence is calculated compared to all sequences in the phylogenic tree.

Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

As used herein, the term “genetic construct” refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.

As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

As used herein, the term “antibody” refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. As used herein, the terms “antibody fragment” or “characteristic portion of an antibody” are used interchangeably and refer to any derivative of an antibody which is less than full-length. In general, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively or additionally, an antibody fragment may comprise multiple chains which are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multimolecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.

As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into an oligonucleotide chain. As used herein, the terms “nucleic acid” and “polynucleotide” can be used interchangeably. In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. The term “nucleic acid sequence” as used herein can refer to the nucleic acid material itself and is not restricted to the sequence information (e.g. the succession of letters chosen, for example, among the five base letters A, G, C, T, or U) that biochemically characterizes a specific nucleic acid, e.g., a DNA or RNA molecule. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. The term “nucleic acid segment” is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In some embodiments, a “nucleic acid” or “polynucleotide” comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

As used herein, a “particle” refers to any entity having a diameter of less than 100 microns (μm). Typically, particles have a longest dimension (e.g. diameter) of 1000 nm or less. In some embodiments, particles have a diameter of 300 nm or less. In some embodiments, nanoparticles have a diameter of 200 nm or less. In some embodiments, nanoparticles have a diameter of 100 nm or less. In general, particles are greater in size than the renal excretion limit, but are small enough to avoid accumulation in the liver. In some embodiments, a population of particles may be relatively uniform in terms of size, shape, and/or composition. In general, inventive particles are biodegradable and/or biocompatible. Inventive particles can be solid or hollow and can comprise one or more layers. In some embodiments, particles are spheres, spheroids, flat, plate-shaped, cubes, cuboids, ovals, ellipses, cylinders, cones, or pyramids. In some embodiments, particles can be a matrix of polymers. In some embodiments, the matrix is cross-linked. In some embodiments, formation of the matrix involves a cross-linking step. In some embodiments, the matrix is not substantially cross-linked. In some embodiments, formation of the matrix does not involve a cross-linking step. In some embodiments, particles can be a non-polymeric particle (e.g. a metal particle, quantum dot, ceramic, inorganic material, bone, etc.). Components of the pharmaceutical compositions and vaccines disclosed herein may be particles or may be microparticles, nanoparticles, liposomes, and/or micelles. As used herein, the term “nanoparticle” refers to any particle having a diameter of less than 1000 nm.

As used herein, the term “subject” or “patient” refers to any organism to which a composition of this invention may be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants. The term “subject” is used throughout the specification to describe an animal to whom treatment with the compositions according to the present invention is provided or administered. For treatment of those conditions which are specific for a specific subject, such as a human being or such as a mammal, the term “patient” may be interchangeably used. In some instances in the description of the present invention, the term “patient” will refer to human patients. In some embodiments, the subject may be a mammal to whom the present invention is provided or administered.

As used herein, the term “targeting moiety” refers to any moiety that binds to a component associated with a cell. Such a component is referred to as a “target” or a “marker.” A targeting moiety may be a polypeptide, glycoprotein, nucleic acid, small molecule, carbohydrate, lipid, etc. In some embodiments, a targeting moiety is an antibody or characteristic portion thereof. In some embodiments, a targeting moiety is a receptor or characteristic portion thereof. In some embodiments, a targeting moiety is a ligand or characteristic portion thereof. In some embodiments, a targeting moiety is a nucleic acid targeting moiety (e.g. an aptamer) that binds to a cell type specific marker. In general, an aptamer is an oligonucleotide (e.g., DNA, RNA, or an analog or derivative thereof) that specifically binds to a particular target, such as a polypeptide. In some embodiments, a targeting moiety is a small molecule.

Compositions

Embodiments according to the disclosure include surfactant vesicles comprising mixtures of oppositely-charged single-tailed surfactants (commonly referred to as “catanionic” vesicles). Compositions of the disclosure comprise one or a plurality of vesicles comprising whole cell extract. In some embodiments, the catanionic vesicles comprised functionalized carbohydrate and/or peptide optionally fused with one or a plurality of linkers and/or peptides, nucleic acid molecules, or agent disclosed herein. A composition comprising a bacterial cell lysate fraction, a bacterial cell membrane-bound fraction, and a plurality of catanionic vesicles wherein the bacterial cell lysate components are encapsulated within said catanionic vesicles, an individual catanioic vesicle comprises a fraction of total bacterial cell lysate components and said plurality of catanionic vesicles collectively comprise total bacterial cell lysate components.

Single-tailed, anionic surfactant can include an amphipathic molecule having from about C₆ to about C₂₀ hydrocarbon tail region and a hydrophilic, polar head group. The head-group on the anionic surfactant can be, for example, sulfonate, sulfate, carboxylate, benzene sulfonate, or phosphate. The single-tailed, cationic surfactant can include an amphipathic molecule having a from about C₆ to about C₂₀ hydrocarbon tail region and a hydrophilic polar head group. The head group on the cationic surfactant can be, for example, a quaternary ammonium group, a sulfonium group, or a phosphonium group.

The size and curvature properties (shape) of catanionic vesicles formed according to embodiments of the invention can vary depending upon factors such as the length of the hydrocarbon tail regions of the constituent surfactants and the nature of the polar head groups. At a common 0.1% bioconjugate-to-surfactant ratio, the bioconjugate can have no observable effect on vesicle shape, size, or stability in aqueous media. In some embodiments, the bioconjugate-to-surfactant ratio in weight/weight is from about 0.1% to about 2%. In some embodiments, the bioconjugate-to-surfactant ratio weight/weight is from about 0.1% to about 3%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 4%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 5%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 6%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 7%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 8%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 9%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 10%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 12%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 0.1% to about 15%. In some embodiments, the bioconjugate-to-surfactant weight/weight ratio is from about 1.0% to about 2%. In some embodiments, the bioconjugate-to-surfactant ratio weight/weight is from about 1.0% to about 3%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 4%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 5%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 6%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 7%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 8%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 9%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 10%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 12%. In some embodiments, the bioconjugate-to-surfactant ratio is from about 1.0% to about 15%.

In some embodiments, the bioconjugate-to-surfactant ratio weight/weight is from about 1.0% to about 3%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 4%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 5%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 6%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 7%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 8%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 9%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 10%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 12%. In some embodiments, the LPS or LOS-to-surfactant ratio is from about 1.0% to about 15%.

The bilayer can include cationic surfactant and anionic surfactant in a molar ratio in a range of from about 9:1 to about 1:9, excluding a molar ratio of about 1:1. For example, the bilayer can include cationic surfactant and anionic surfactant in a molar ratio of from about 9:1, 8:2, 7:3, 6:4, 5.5:4.5, 5.1:4.9, 4.9:5.1, 4.5:5.5, 4:6, 3:7, 2:8, and 1:9 to about 9:1, 8:2, 7:3, 6:4, 5.5:4.5, 5.1:4.9, 4.9:5.1, 4.5:5.5, 4:6, 3:7, 2:8, and 1:9. For example, the bilayer can include cationic surfactant and anionic surfactant in a molar ratio in a range of from about 6:4 to about 8:2, in a range of from about 6:4 to about 7:3, of about 6:4, in a range of from about 2:8 to about 4:6, in a range of from about 3:7 to about 4:6, and of about 4:6. The cationic surfactant and the anionic surfactant can have a concentration in the external aqueous environment of less than about 5 wt %. For example, the cationic surfactant and the anionic surfactant can have a concentration in the external aqueous environment of from about 0.0001 wt % to about 3 wt %, for example, of from about 0.5 wt % to about 2 wt %, for example, of about 1 wt %. The solute ion can be present in the aqueous environment at an external concentration, the solute ion can be present in the vesicle at a sequestration concentration, and the ratio of the sequestration concentration to the external concentration can be greater than 1, for example, greater than or equal to 5. For example, from about 20% to about 75% of the solute ion present in the aqueous environment and in the catanionic surfactant vesicle can be sequestered in the catanionic surfactant vesicle. The encapsulation efficiency of the solute ion in the vesicle can be at least about 2%, for example, at least about 3%, greater than about 7%, or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%. The percentage of solute adsorbed on the bilayer can be at least about 0.5%, for example, at least about 1%, 2%, 5%, or 16%. The ratio of the percentage of solute adsorbed on the bilayer to the encapsulation efficiency can be at least about 10%, for example, greater than 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.

The release of solute ion from a catanionic vesicle according to the present invention can occur over a range of time such that the half-life time of the release is from about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 100, 120, 150, 200, and 500 days to about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 100, 120, 150, 200, and 500 days.

The diameter of vesicles according to the disclosure can be, for example from about 10 to about 250 nanometers, for example, from about 30 to about 150 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 20 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 30 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 40 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 50 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 60 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 70 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 80 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 90 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 100 nm to about 200 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 20 nm to about 100 nm. In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a plurality of vesicles with a distribution of vesicles diameter from about 20 nm to about 150 nm.

In some embodiments, the catanionic vesicles or accompanying particles may optionally comprise one or more carbohydrates. The percent of carbohydrate in particles can range from 0% to 99% by weight, from 10% to 99% by weight, from 25% to 99% by weight, from 50% to 99% by weight, or from 75% to 99% by weight. In some embodiments, the percent of carbohydrate in particles can range from 0% to 75% by weight, from 0% to 50% by weight, from 0% to 25% by weight, or from 0% to 10% by weight. In some embodiments, the percent of carbohydrate in the vesicles or particles can be from about 0.05% to about 5.0% by weight. in some embodiments, the percent of carbohydrate in the vesicles or particles can be approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, or approximately 1% by weight, approximately 2% by weight, approximately 3% by weight, approximately 4% by weight, approximately 5% by weight, approximately 10% by weight, approximately 15% by weight, approximately 20% by weight, approximately 25% by weight, or approximately 30% by weight. Carbohydrates may be natural or synthetic. A carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate is a monosaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, a carbohydrate is a disaccharide, including but not limited to lactose, sucrose, maltose, trehalose, and cellobiose. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin, heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In certain embodiments, the carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol. In certain embodiments, the carbohydrate is a bacterial carbohydrate. In certain embodiments, the carbohydrate is a bacterial carbohydrate derived directly from the whole cell extract from which the catanionic vesicle is made. In certain embodiments, the carbohydrate is LOS or LPS. In certain embodiments, the carbohydrate is LOS or LPS and derived directly from the whole cell extract from which the catanionic vesicle is made.

In some embodiments, the catanionic vesicles, vaccines or accompanying particles may optionally comprise one or a plurality of bacterial lipoproteins. The lipoproteins may be any lipoprotein disclosed in Table 1 (identified by UniProt number and incorporated by reference in its entirety), either individually or in combination, and/or antigenic fragments that 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homolgous to the lipoproteins identified by UniProt number. In some embodiments, the catanionic vesicles or accompanying particles may optionally comprise one or any combination of amino acids that are 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homolgous to SEQ ID NO:1 through SEQ ID NO:883. In some embodiments, the catanionic vesicles, vaccines or accompanying particles may optionally comprise one or a plurality of bacterial antigens identified by UniProt number in Table 1 and/or antigenic fragments that 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homolgous to the bacterial antigens identified by UniProt number in Table 1. In some embodiments, the catanionic vesicles, vaccines or accompanying particles may optionally comprise one or a plurality of Francisella lipoproteins or Francisella antigens. Examples of Francisella antigens of the present disclosure may be DnaK, Tul4, and those antigens disclosed in Example 4.

Vesicle size can be influenced by selecting the relative lengths of the hydrocarbon tail regions of the anionic and cationic surfactants. For example, large vesicles, e.g., vesicles of from 150 to 200 nanometers diameter, can be formed when there is disparity between the length of the hydrocarbon tail on the anionic surfactant and the hydrocarbon tail on the cationic surfactant. For example, large vesicles can be formed when a C₁₆ cationic surfactant solution is combined with a C₈ anionic surfactant solution. Smaller vesicles can be produced by using anionic and cationic surfactant species of which the lengths of the hydrocarbon tails are more closely matched. The permeability characteristics of vesicles according to the present invention can be influenced by the nature of the constituent surfactants, for example, the chain length of the hydrocarbon tail regions of the surfactants. Longer tail lengths on the surfactant molecules can decrease the permeability of the vesicles by increasing the thickness and hydrophobicity of the vesicle membrane (bilayer). The control of reagent and substrate permeation across vesicle membranes can be an important parameter, for example, when using the vesicles as microreactors.

Exemplary anionic, single-chain surface active agents include alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, and saturated or unsaturated fatty acids and their salts. Moieties comprising the polar head group in the cationic surfactant can include, for example, quaternary ammonium, pyridinium, sulfonium, and/or phosphonium groups. For example, the polar head group can include trimethylammonium. Exemplary cationic, single-chain surface active agents include alkyl trimethylammonium halides, alkyl trimethylammonium tosylates, and N-alkyl pyridinium halides.

Alkyl sulfates can include sodium octyl sulfate, sodium decyl sulfate, sodium dodecyl sulfate, and sodium tetra-decyl sulfate. Alkyl sulfonates can include sodium octyl sulfonate, sodium decyl sulfonate, and sodium dodecyl sulfonate. Alkyl benzene sulfonates can include sodium octyl benzene sulfonate, sodium decyl benzene sulfonate, and sodium dodecyl benzene sulfonate (SDBS). Fatty acid salts can include sodium octanoate, sodium decanoate, sodium dodecanoate, and the sodium salt of oleic acid.

Alkyl trimethylammonium halides can include octyl trimethylammonium bromide, decyl trimethylammonium bromide (DTAB), dodecyl trimethylammonium bromide, myristyl trimethylammonium bromide, and cetyl trimethylammonium bromide. Alkyl trimethylammonium tosylates can include octyl trimethylammonium tosylate, decyl trimethylammonium tosylate, dodecyl trimethylammonium tosylate, myristyl trimethylammonium tosylate, and cetyl trimethylammonium tosylate. For example, N-alkyl pyridinium halides can include decyl pyridinium chloride (DPC), dodecyl pyridinium chloride, cetyl pyridinium chloride, decyl pyridinium bromide, dodecyl pyridinium bromide, cetyl pyridinium bromide, decyl pyridinium iodide, dodecyl pyridinium iodide, cetyl pyridinium iodide.

Surfactants that can be used to form catanionic vesicles according to the present invention include, for example, sodium didoceyl sulfate (SDS), Dodecyltrimethylammonium chloride (DTAC), dodecyl(trimethyl)azanium bromide (DTAB), dodecyl-phosphatidylcholine (DPC), Decyl Dimethyl Amine Oxide (DDAO), dodecyldimethyl ammonium bromide (DDAB), sodium octylsulfate (SOS), dioctyl sulfosuccinate sodium salt (AOT), and cetyltrimethylammonium tosylate (CTAT). In some embodiments, the vesicles comprise bioconjugates, for example, glycoconjugates, such as alkylated carbohydrates. In some embodiments, these vesicles can sequester and separate charged biomolecules in solution. To add increased biofunctionality to these vesicles, or to target the delivery of sequestered molecules, these catanionic vesicles can be enhanced with the addition of one or more bioconjugates, both charged and non-ionic, in order to interact with natural or artificial carbohydrate and/or protein recognition systems. In some embodiments, these carbohydrate- and/or protein-functionalized vesicles present a protein and/or sugar moeity to an actual cell surface and facilitate multivalent interactions. The recognition process for a carbohydrate is fundamentally different than protein-protein or antibody-antigen interactions at cell surfaces in that carbohydrate recognition is a multivalent process. Because each binding event of a carbohydrate-mediated system involves weak interactions (H-bonding), then the receptors involved must establish multiple interactions to achieve high selectivity (Mammen, S. K. Choi and G. M. Whitesides, Angew. Chem. Int. Ed., 1998, 37, 2755-2794). Accordingly, the recognition of glycosyl residues on the cell surface requires clustering or a high density of surface receptors. It is this multivalent binding process of oligosaccharide-mediated recognition that can in certain cases be advantageous in comparison with recognition strategies associated with other biomolecules such as proteins or nucleic acids.

Aspects of the disclosure relate to a composition comprising: a catanionic surfactant vesicle; and at least one bacterial immunogen; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants; wherein the at least one bacterial immunogen comprises at least one membrane-bound sugar, a polypeptide, a functional fragment thereof, a combination thereof from the membrane of a bacterial cell wall; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is embedded at least partially in the bilayer; and wherein the catanionic surfactant vesicle comprises at least about 50 micrograms per microliter of bacterial oligosaccharide or polysaccharide.

The present disclosure also relates a composition comprising: a catanionic surfactant vesicle; and at least one bacterial immunogen; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants; wherein the at least one bacterial immunogen from the membrane of a bacterial cell wall comprises at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the bilayer; and wherein the bacterial immunogen is chosen from one or a combination of: DnaK, Tul4, FopA. In some embodiments, the catanionic surfactant vesicle comprises a combination of DnaK and Tul4.

The present disclosure also relates a composition comprising: a catanionic surfactant vesicle; and at least one bacterial immunogen; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants; wherein the at least one bacterial immunogen from the membrane of a bacterial cell wall comprises at least one membrane-bound sugar, polypeptide, functional fragment thereof, a combination thereof; and wherein the at least one sugar, polypeptide, functional fragment thereof, or combination thereof is positioned at least partially in the bilayer.

In some embodiments, particles may optionally comprise one or more carbohydrates. The percent of carbohydrate in particles can range from 0% to 99% by weight, from 10% to 99% by weight, from 25% to 99% by weight, from 50% to 99% by weight, or from 75% to 99% by weight. In some embodiments, the percent of carbohydrate in particles can range from 0% to 75% by weight, from 0% to 50% by weight, from 0% to 25% by weight, or from 0% to 10% by weight. In some embodiments, the percent of carbohydrate in particles can be approximately 1% by weight, approximately 2% by weight, approximately 3% by weight, approximately 4% by weight, approximately 5% by weight, approximately 10% by weight, approximately 15% by weight, approximately 20% by weight, approximately 25% by weight, or approximately 30% by weight. Carbohydrates may be natural or synthetic. A carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate is a monosaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, and neuramic acid. In certain embodiments, a carbohydrate is a disaccharide, including but not limited to lactose, sucrose, maltose, trehalose, and cellobiose. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon, amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin, heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In certain embodiments, the carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol. In certain embodiments, a carbohydrate is LOS or LPS.

Aspects of the disclosure relate to pharmaceutical compositions comprising any one or plurality of vesicles disclosed herein and at least one pharmaceutically acceptable carrier or excipient. The present invention provides novel targeted particles comprising: a therapeutically effective amount of a particle, one or more targeting moieties (e.g. aptamers), and one or more therapeutic agents to be delivered; and one or more pharmaceutically acceptable excipients. In some embodiments, the present invention provides for pharmaceutical compositions comprising inventive targeted particles as described herein. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically-active substances. In accordance with some embodiments, a method of administering a pharmaceutical composition comprising inventive compositions to a subject in need thereof is provided. In some embodiments, inventive compositions are administered to humans. For the purposes of the present invention, the phrase “active ingredient” generally refers to a vesicle disclosed herein and one or more therapeutic agents to be delivered.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with one or more excipients and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable excipient(s), and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between about 0.1% to about 99% (w/w) active ingredient.

Pharmaceutical formulations of the present invention may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

In some embodiments, the pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, the excipient is approved for use in humans and for veterinary use. In some embodiments, the excipient is approved by United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the inventive formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents can be present in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.

Exemplary vaccine adjuvants include, but are not limited to, aluminium phosphate, aluminium hydroxide, calcium phosphate, monophosphoryl lipid A (MPL), Quil A/QS-21, inulin-derivatives, bacterial peptidoglycan, CpG-motif containing DNA, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), trehalose dimycolate (TDM).

Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfate, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the targeted particles of the invention are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof. In some embodiments, the liquid dosage form is an aqueous solution or water. In some embodiments, the liquids dosage form heated and/or sterilized water.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the targeted particles of this invention with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

The active ingredients can be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a targeted particle of this invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active component is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate may be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.

In some embodiments, the pharmaceutical compositions may further comprise one or a plurality of agents. According to the present invention, pharmaceutical compositions comprising any one or plurality of vesicles disclosed herein may be used for delivery of any agent, including, for example, therapeutic, diagnostic, and/or prophylactic agents. Exemplary agents to be delivered in accordance with the present invention include, but are not limited to, small molecules, organometallic compounds, nucleic acids, proteins (including multimeric proteins, protein complexes, etc.), peptides, lipids, carbohydrates, hormones, metals, radioactive elements and compounds, drugs, vaccines, immunological agents, etc., and/or combinations thereof.

In some embodiments, pharmaceutical compositions comprising any one or plurality of vesicles disclosed herein comprise less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, less than 1% by weight, or less than 0.5% by weight of the therapeutic agent to be delivered.

In some embodiments, the agent to be delivered may be a mixture of pharmaceutically active agents. For example, a local anesthetic may be delivered in combination with an anti-inflammatory agent such as a steroid. To give but another example, an antibiotic may be combined with an inhibitor of the enzyme commonly produced by bacteria to inactivate the antibiotic (e.g., penicillin and clavulanic acid).

In some embodiments, the agent to be delivered may be a mixture of anti-cancer agents. In some embodiments, inventive targeted particles are administered in combination with one or more of the anti-cancer agents described herein. To give but one example, in some embodiments, inventive compositions comprising an anti-cancer agent to be delivered are administered in combination with hormonal therapy. The growth of some types of tumors can be inhibited by providing or blocking certain hormones. For example, steroids (e.g. dexamethasone) can inhibit tumor growth or associated edema and may cause regression of lymph node malignancies. In some cases, prostate cancer is often sensitive to finasteride, an agent that blocks the peripheral conversion of testosterone to dihydrotestosterone. Breast cancer cells often highly express the estrogen and/or progesterone receptor. Inhibiting the production (e.g. with aromatase inhibitors) or function (e.g. with tamoxifen) of these hormones can often be used in breast cancer treatments. In some embodiments, gonadotropin-releasing hormone agonists (GnRH), such as goserelin possess a paradoxic negative feedback effect followed by inhibition of the release of follicle stimulating hormone (FSH) and leuteinizing hormone (LH), when given continuously.

In some embodiments, the agent to be delivered may be a mixture of Cas9 enzyme linked to an RNA molecule and a DNA template. Such a payload can be designed to repair single base pair mutations in genomic DNA of a subject.

In some embodiments, the agent to be delivered is a small molecule and/or organic compound with pharmaceutical activity. In some embodiments, the agent is a clinically-used drug. In some embodiments, the drug is an anti-cancer agent, antibiotic, anti-viral agent, anti-HIV agent, anti-parasite agent, anti-protozoal agent, anesthetic, anticoagulant, inhibitor of an enzyme, steroidal agent, steroidal or non-steroidal anti-inflammatory agent, antihistamine, immunosuppressant agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, sedative, opioid, analgesic, anti-pyretic, birth control agent, hormone, prostaglandin, progestational agent, anti-glaucoma agent, ophthalmic agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, neurotoxin, hypnotic, tranquilizer, anti-convulsant, muscle relaxant, anti-Parkinson agent, anti-spasmodic, muscle contractant, channel blocker, miotic agent, anti-secretory agent, anti-thrombotic agent, anticoagulant, anti-cholinergic, .beta.-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, vasodilating agent, anti-hypertensive agent, angiogenic agent, modulators of cell-extracellular matrix interactions (e.g. cell growth inhibitors and anti-adhesion molecules), inhibitors of DNA, RNA, or protein synthesis, etc.

In certain embodiments, the therapeutic agent to be delivered is an anti-cancer agent (i.e. cytotoxic agents). Most anti-cancer agents can be divided in to the following categories: alkylating agents, antimetabolites, natural products, and hormones and antagonists. Anti-cancer agents typically affect cell division and/or DNA synthesis. However, some chemotherapeutic agents do not directly interfere with DNA. To give but one example, tyrosine kinase inhibitors (imatinib mesylate/Gleevec™) directly target a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors, etc.). Alkylating agents are so named because of their ability to add alkyl groups to many electronegative groups under conditions present in cells. Alkylating agents typically function by chemically modifying cellular DNA. Exemplary alkylating agents include nitrogen mustards (e.g. mechlorethamine, cyclophosphamide, ifosfamide, melphalan (1-sarcolysin), chlorambucil), ethylenimines and methylmelamines (e.g. altretamine (hexamethylmelamine; HMM), thiotepa (triethylene thiophosphoramide), triethylenemelamine (TEM)), alkyl sulfonates (e.g. busulfan), nitrosureas (e.g. carmustine (BCNU), lomustine (CCMU), semustine (methyl-CCNU), streptozocin (streptozotocin)), and triazenes (e.g. dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)). Antimetabolites act by mimicking small molecule metabolites (e.g. folic acid, pyrimidines, and purines) in order to be incorporated into newly synthesized cellular DNA. Such agents also affect RNA synthesis. An exemplary folic acid analog is methotrexate (amethopterin). Exemplary pyrimidine analogs include fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside). Exemplary purine analogs include mercaptopurine (6-mercaptopurine; 6-MP), azathioprine, thioguanine (6-thioguanine; TG), fludarabine phosphate, pentostatin (2′-deoxycoformycin), cladribine (2-chlorodeoxyadenosine; 2-CdA), and erythrohydroxynonyladenine (EHNA). Natural small molecule products which can be used as anti-cancer agents include plant alkaloids and antibiotics. Plant alkaloids and terpenoids (e.g. vinca alkaloids, podophyllotoxin, taxanes, etc.) typically block cell division by preventing microtubule function. Vinca alkaloids (e.g. vincristine, vinblastine (VLB), vinorelbine, vindesine, etc.) bind to tubulin and inhibit assembly of tubulin into microtubules. Vinca alkaloids are derived from the Madagascar periwinkle, Catharanthus roseus (formerly known as Vinca rosea). Podophyllotoxin is a plant-derived compound used to produce two other cytostatic therapeutic agents, etoposide and teniposide, which prevent cells from entering the G1 and S phases of the cell cycle. Podophyllotoxin is primarily obtained from the American Mayapple (Podophyllum peltatum) and a Himalayan Mayapple (Podophyllum hexandrum). Taxanes (e.g. paclitaxel, docetaxel, etc.) are derived from the Yew Tree. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase. Antibiotics which can be used as anti-cancer agents include dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, idarubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mytomycin C).

Other small molecules which can be used as anti-cancer agents include platinum coordination complexes (e.g. cisplatin (cis-DDP), carboplatin), anthracenedione (e.g. mitoxantrone), substituted urea (e.g. hydroxyurea), methylhydrazine derivatives (e.g. procarbazine (N-methylhydrazine, MIH), and adrenocortical suppressants (e.g. mitotane (o,p′-DDD), aminoglutethimide).

Hormones which can be used as anti-cancer agents include adrenocorticosteroids (e.g. prednisone), aminoglutethimide, progestins (e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate), estrogens (e.g. diethylstilbestrol, ethinyl estradiol), antiestrogen (e.g. tamoxifen), androgens (e.g. testosterone propionate, fluoxymesterone), antiandrogens (e.g. flutamide), and gonadotropin-releasing hormone analog (e.g. leuprolide).

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 μm to about 0.7 μm or from about 1 μm to about 6 μm. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 μm and at least 95% of the particles by number have a diameter less than 7 μm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 μm and at least 90% of the particles by number have a diameter less than 6 μm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65 degrees F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 μm to about 200 μm.

The formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.

General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005.

Vaccines

The present disclosure relates to pharmaceutical compositions comprising vaccines that comprise one or any combination of vesicles disclosed herein and a pharmaceutically acceptable carrier. Vesicles comprising any one or more glycocongugates, glycolipids, lipids, sugars, DNA, RNA, proteins, and glycoproteins disclosed herein can comprise an antigen itself and/or an adjuvant. In some embodiments, the pharmaceutical composition is a vaccine designed to elicit an antigen-specific immune response against a pathogen or an antigen associated with a hyperproliferative disease, such as cancer. In some embodiments, the vaccine comprises a vesicle comprising an antigen derived from a Gram-negative bacterial cell. In some embodiments, the vaccine comprises an antigen derived directly from or extracted directly from a Gram-negative bacterial cell. In some embodiments, the Gram-negative bacterial cell is a Francisella or Neisseria bacterial cell. In some embodiments, the Gram-negative bacterial cell is derived from or is a Francisella tularensis bacterial cell. In some embodiments, the Gram-negative bacterial cell is derived from or is a Neisseria gonorrhoeae bacterial cell. In some embodiments, the Gram-negative bacterial cell is derived from or is a Neisseria meningitidis bacterial cell. In some embodiments, the Gram-negative bacterial cell is a Pseudomonas bacterial cell. In some embodiments, the Gram-negative bacterial cell is a Pseudomonas aeruginosa bacterial cell. The present disclosure relates to pharmaceutical compositions comprising vaccines that comprise one or any combination of vesicles disclosed herein and a pharmaceutically acceptable carrier for the treatment and or prevention of bacterial infection. In some embodiments, at least one antigen elicting the immune response is contained at least partially in the leaflet (or bilayer) of the vesicle.

Vaccines are disclosed which arise from a multi-phase strategy to enhance cellular immune responses induced by immunogens. The novel construct has been designed to elicit stronger and broader cellular immune responses than a corresponding codon optimized immunogens.

The vaccines are based upon proteins and genetic constructs that encode proteins with epitopes that make them particularly effective as immunogens against which bacterial antigens can be induced. Accordingly, vaccines may induce a therapeutic or prophylactic immune response. In some embodiments, the means to deliver the immunogen is a catanionic vesicle comprising a protein subunit, a composition comprising the catanionic vesicle and attenuated vaccine, killed vaccine, nucleic acid, or any of the components of adjuvants disclosed herein. In some embodiments, the vaccine comprises a combination selected from the groups consisting of: one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions comprising the immunogen, one or more attenuated vaccines and one or more killed vaccines.

According to some embodiments, a vaccine is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response against bacteria or an antigen. In some embodiments the bacteria or antigen from a bacterial cell is from or derived from at least one Gram-negative bacterial cell. Compositions and methods are provided which prophylactically and/or therapeutically immunize an individual against Gram-negative bacteria. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and virus, genetically altered organisms or viruses, and cell extracts. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents may include antigens of such bacterial organisms as Streptococcus pnuemoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtherias, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens of such viruses as smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and the like; antigens of fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. In some embodiments, the vaccine does not comprise a killed organism. In some embodiments, the vaccine does not comprise a killed organism that is a Gram-negative bacterial cell such as from the genus Neisseria or Francisella.

Compositions for delivery of catanionic vesicles that comprise at least one bacterial immunogen can comprise a genetic construct with a coding sequence operably linked to regulatory elements. Compositions may include a plasmid that encodes an immunogen, a recombinant vaccine comprising a nucleotide sequence that encodes the immunogen, a live attenuated pathogen that encodes a protein of the invention and/or includes a protein of the invention; a killed pathogen includes a protein of the invention; or a composition such as a catanionic vesicle or subunit vaccine that comprises a protein of the invention. The present invention further relates to injectable pharmaceutical compositions that comprise compositions disclosed herein.

In some embodiments, vaccines include any bacterial immunogen identified on Table 1 or functional fragments thereof. In some embodiments, antigenic fragment are those fragments SEQ ID NOs: 1 through 883. In some embodiments, vesicles, pharmaceutical compositions, and/or vaccines comprise any bacterial immunogen identified on Table 1 or antigenic fragments thereof. In some embodiments, vesicles, pharmaceutical compositions, and/or vaccines comprise any one or combination of: SEQ ID No: 1 through SEQ ID No: 883 disclosed or antigenic fragments thereof that are 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% homologous to SEQ ID No: 1 through SEQ ID No: 883. In some embodiments, vesicles, pharmaceutical compositions, and/or vaccines comprise a Neisseria antigen or antigenic fragments thereof that are 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% homologous to a Neisseria antigen. Neisseria antigens can be found on Table 1 and any one or combination can be a component of the disclosed vesicles, pharmaceutical compositions, and/or vaccines. In some embodiments, the vaccine is a multivalent vaccine comprising a catanionic vesicle comprising whole cell extract with from about 1 pg to about 1 microgram of LPS or LOS and at least one or a plurality of lipoproteins and/or membrane-bound proteins taken from the whole cell extract from which the vesciles are formed.

Vaccines of the disclosure can comprise vesicles and one or a plurality of additional components that enhance an immune response stimulated by the vaccine. For instance vaccines of the present disclosure can comprise a nucleic acid molecule that encodes one or a plurality of immunomodulating proteins or antigens. In some embodiments, vaccines of the disclosure comprise proteins and genetic constructs that encode proteins with epitopes that make them particularly effective as immunogens against which bacterial immune responses can be induced. Accordingly, vaccines can be provided to induce a therapeutic or prophylactic immune response against a pathogen antigen or cancer-related antigen. In some embodiments, vaccines can be provided to induce a therapeutic or prophylactic immune response against a bacterial antigen such as a Gram-negative bacterial cell or Gram negative. In some embodiments, the means to deliver the immunogen is a vesicle comprising or in combination with a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, a composition comprising a catanionic vesicle, an attenuated pathogen or a killed pathogen. In some embodiments, the vaccine comprises a combination selected from the groups consisting of: one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions comprising the immunogen, one or more catanionic vesicles disclosed herein, one or more attenuated vaccines and one or more killed vaccines.

Aspects of the invention provide methods of delivering the coding sequences of the protein on nucleic acid molecule such as plasmid, as part of recombinant vaccines and as part of attenuated vaccines, as isolated proteins or proteins part of a vector.

According to some aspects of the present invention, compositions and methods are provided which prophylactically and/or therapeutically immunize subject.

DNA vaccines are described in U.S. Pat. Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and the priority applications cited therein, which are each incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are both incorporated herein by reference.

The present invention relates to compositions comprising improved attenuated live vaccines, improved killed vaccines and improved vaccines that use recombinant vectors to deliver foreign genes that encode antigens and well as subunit and glycoprotein vaccines in combination with a catanionic vesicle disclosed herein. Examples of attenuated live vaccines, those using recombinant vectors to deliver foreign antigens, subunit vaccines and glycoprotein vaccines are described in U.S. Pat. Nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are each incorporated herein by reference in their entireties.

When taken up by a cell, a vesicle or vaccine of the present disclosure may fuse with a cell and distribute its payload. If the vaccine comprises a vesicle with a genetic construct, payload of the vesicle may remain present in the cell and stimulate an immune response, and, in the case of nucleic acid, may express as a functioning extra chromosomal molecule and/or integrate into the cell's chromosomal DNA. DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids. Alternatively, linear DNA that can integrate into the chromosome may be introduced into the cell. When introducing DNA into the cell, reagents that promote DNA integration into chromosomes may be added. DNA sequences that are useful to promote integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Gene constructs may remain part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. Gene constructs may be part of genomes of recombinant viral vaccines where the genetic material either integrates into the chromosome of the cell or remains extrachromosomal. Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of the sequence that encodes the target protein or the immunomodulating protein. It is necessary that these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in the individual to whom they are administered.

Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within the cells of the individual.

Examples of promoters useful to include on a given genetic construct, especially in the production of a genetic vaccine for mammals including humans, include but are not limited to promoters from Simian Virus 40 (SV 40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MY) such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to SV 40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV 40 polyadenylation signal that is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids such as pVAX from Invitrogen (San Diego, Calif.) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration.

In some embodiments, vesicle(s) are delivered which include or comprise nucleotide sequences that encode protein, and, additionally, genes for proteins which further enhance the immune response against such target proteins. Examples of such genes are those which encode other cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFa, TNF˜, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86 and IL-15 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which may be useful include those encoding: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAILR3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

An additional element may be added which serves as a target for cell destruction if it is desirable to eliminate cells receiving the genetic construct for any reason. A herpes thymidine kinase (tk) gene in an expressible form can be included in the genetic construct. The drug gangcyclovir can be administered to the individual and that drug will cause the selective killing of any cell producing tk, thus, providing the means for the selective destruction of cells with the genetic construct.

In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a vesicle with a targeting moiety. In some embodiments, vesicle can comprise a matrix of polymers. In some embodiments, a therapeutic agent to be delivered and/or targeting moiety can be associated with the surface of, encapsulated within, surrounded by, and/or dispersed throughout the bilayer of the vesicle.

In some embodiments, a polymeric matrix can comprise polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and/or polyamines. In some embodiments, a polymeric matrix may comprise poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and/or copolymers thereof. In some embodiments, a polymeric matrix can comprise dendrimers, proteins, carbohydrates, and/or nucleic acids.

In some embodiments, the compositions, pharmaceutical compositions, and vaccines disclosed herein comprise a vesicle and a particle, such as a non-polymeric particles (e.g. metal particles, quantum dots, ceramics, inorganic materials, bone, etc.), which in combination stimulate the immune system to a magnitude sufficient to treat or prevent infection of a pathogen or a hyperproliferative disorder. In some embodiments, a therapeutic agent and/or targeting moiety can be covalently associated with a molecule in the vesicle. In some embodiments, a therapeutic agent and/or targeting moiety can be non-covalently associated with a non-polymeric particle or the vesicle disclosed herein. In some embodiments, a therapeutic agent and/or targeting moiety can be associated with the surface of, encapsulated within, surrounded by, and/or dispersed throughout a non-polymeric polymer or the vesicle(s) disclosed herein. In some embodiments, the vesicles and/or particles may optionally comprise one or more sugars, lipids, or release-retarding ingredients.

In certain embodiments, targeted particles in accordance with the present invention comprise a targeting moiety which specifically binds to one or more targets associated with an organ, tissue, cell, extracellular matrix, and/or intracellular compartment. As used herein, the terms “target” and “marker” can be used interchangeably.

A targeting moiety may be a nucleic acid (e.g. aptamer), polypeptide (e.g. antibody), glycoprotein, small molecule, carbohydrate, lipid, etc. For example, a targeting moiety can be an aptamer, which is generally an oligonucleotide (e.g., DNA, RNA, or an analog or derivative thereof) that binds to a particular target, such as a polypeptide. In general, the targeting function of the aptamer is based on the three-dimensional structure of the aptamer. In some embodiments, a targeting moiety is a polypeptide (e.g. an antibody that specifically recognizes a tumor marker).

In some embodiments, a target may be a marker that is exclusively or primarily associated with one or a few tissue types, with one or a few cell types, with one or a few diseases, and/or with one or a few developmental stages. In some embodiments, a target can comprise a protein (e.g. cell surface receptor, transmembrane protein, etc.), a carbohydrate (e.g. glycan moiety, glycocalyx, etc.), a lipid (e.g. steroid, phospholipid, etc.), and/or a nucleic acid (e.g. DNA, RNA, etc.)

In some embodiments, a target (i.e. marker) is a molecule that is present exclusively or in higher amounts on a neoplastic or malignant cell, e.g., a tumor antigen. In some embodiments, a marker is a prostate cancer marker. In certain embodiments, the prostate cancer marker is prostate specific membrane antigen (PSMA), a 100 kDa transmembrane glycoprotein that is expressed in most prostatic tissues, but is more highly expressed in prostatic cancer tissue than in normal tissue.

The present invention provides methods for designing novel targeting moieties. The present invention further provides methods for isolating or identifying novel targeting moieties from a mixture of candidate targeting moieties. Nucleic acid targeting moieties (e.g. aptamers) may be designed and/or identified using any available method, including SELEX and PICO, as described herein.

According to the present invention, any agents, including, for example, therapeutic agents (e.g. anti-cancer agents), diagnostic agents (e.g. contrast agents; radionuclides; and fluorescent, luminescent, and magnetic moieties), prophylactic agents (e.g. vaccines), and/or nutraceutical agents (e.g. vitamins, minerals, etc.) may be delivered and the vesicles disclosed herein may comprise any one or combination of agents. Exemplary agents to be delivered in accordance with the present invention include, but are not limited to, small molecules (e.g. cytotoxic agents), nucleic acids (e.g. RNAi agents), proteins (e.g. antibodies), lipids, carbohydrates, hormones, metals, radioactive elements and compounds, drugs, vaccines, immunological agents, etc., and/or combinations thereof. In some embodiments, the agent to be delivered is an agent useful in the treatment of cancer (e.g. breast cancer).

In some embodiments, the agent to be delivered may be a mixture of pharmaceutically active agents. In some embodiments, the agent to be delivered may be a mixture of anti-cancer agents. In some embodiments, inventive targeted particles are administered in combination with one or more of the anti-cancer agents described herein.

In some embodiments, targeting moieties and/or therapeutic agents are covalently associated with a particle, and release and delivery of the therapeutic agent to a target site occurs by disrupting the association. In some embodiments, targeting moieties and/or therapeutic agents are not covalently associated with a particle. For example, particles may comprise a polymeric matrix, and therapeutic agents may be associated with the surface of, encapsulated within, and/or distributed throughout the polymeric matrix. Therapeutic agents can be released by diffusion, degradation of the particle, and/or combination thereof.

In some embodiments, the vaccine comprises a catanionic vesicle comprising whole bacterial cell extract and comprises LPS, LOS or other sugar molecule derived directly from the bacterial cell from which it is made. Further embodiments comprise one or a plurality of lipoproteins that derived directly from the bacterial cell from which the vesicle is made.

Methods

To increase the likelihood that molecules including glycolipids and cell membrane proteins are incorporated into vesicles during preparation, the disclosure relates to methods of extracting membrane-bound and/or cell wall-bound proteins and glycolipids directly from cells without a purification step by exposing surfactants and compositions comprising surfactants disclosed herein to whole cells or whole cell extract. The cells may be any cells including, for example, bacterial cells, mammalian cells, or insect cells. By forming spontaneous vesicles with whole cells, vesicles can capture membrane-bound forms of glycolipids and cell membrane proteins in the leaftlet of the vesicle and they capture solute and their respective cytosolic fractions in the lumens. By adjusting the exposure time of the vesicles to various components of the method steps, more or less membrane-bound glycolipids and cell membrane proteins can be loaded into the bilayer of the vesicles.

Aspects of the invention relate to methods of extracting membrane-bound fractions of cells at high loading efficiencies to increase the number or concentration of cytosolic or membrane-bound proteins loaded onto the vesicles. Aspects of the invention also relate to

Some embodiments of the disclosure relate to a method of reducing or masking the immune response stimulated by a bacterial sugar molecule by exposing the bacterial sugar to any of the steps to formulate the vesicles disclosed herein.

The disclosure relates to a method of integrating, or loading, a bacterial antigen into a vesicle, comprising: (a) centrifuging a bacterial culture in a bacterial growth medium; (b) removing the bacterial growth medium; (c) adding a weight/weight molar ratio of cationic surfactant to anionic from about 80:20 and or 20:80 in aqueous solution to cellular debris; (d) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d). In some embodiments, the aqueous solution is water. In some embodiments, the disclosure relates to a method of creating a vaccine containing bacterial antigen, comprising: of integrating, or loading, a bacterial antigen into a vesicle, comprising: (a) centrifuging a bacterial culture in a bacterial growth medium; (b) removing the bacterial growth medium; (c) adding a weight/weight molar ratio of cationic surfactant to anionic from about 80:20 and or 20:80 in about ten milliliters of water to bacterial culture; (d) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d). In some embodiments, the aqueous solution is water. In some embodiments, the disclosure relates to a method of creating a vaccine containing bacterial antigen, comprising: of integrating, or loading, a bacterial antigen into a vesicle, comprising: (a) centrifuging a bacterial culture in a bacterial growth medium; (b) removing the bacterial growth medium; (c) adding a weight/weight molar ratio of cationic surfactant to anionic of about 30:70 CTAT:SDBS in about aqueous solution to bacterial culture; (d) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d).

The disclosure provides a method of integrating a bacterial antigen into a vesicle, comprising: (a) centrifuging a bacterial culture in a bacterial growth medium; (b) removing the bacterial growth medium; (c) adding about 70 mg SDBS and in about 9.9 mL of aqueous solution to cellular debris; (d) stirring the mixture at room temperature for at least 30 minutes; (d) adding about 30 mg CTAT in solution to resulting mixture; and (e) stirring the mixture at room temperature for at least about 30 minutes.

The disclosure provides a method of integrating a bacterial antigen into a vesicle, comprising: (a) pelleting bacteria culture by centrifugation; (b) adding about 30 mg CTAT and about 9.9 mL H20 to cellular debris to form an aqueous mixture; (c) stirring the aqueous mixture at room temperature for at least about 30 minutes; (d) adding 70 mg SDBS in solution to the aqueous mixture; and (e) stirring the resulting mixture at room temperature for at least 30 minutes.

The disclosure provides a method of integrating a bacterial antigen into a vesicle, comprising:

(a) pelleting bacteria culture by centrifugation and removing any bacterial culture medium;

(b) adding about 70 mg SDBS and about 9.9 mL water to form an aqueous mixture;

(c) stirring the aqueous mixture at room temperature for at least about 30 minutes;

(d) adding about 30 mg of solid CTAT to the aqueous mixture; and

(e) stirring the aqueous mixture at room temperature for at least about 30 minutes.

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL from about 7 to about 8:1 comprising: (a) centrifuging bacterial cells; (b) removing any bacterial growth medium; (c) adding a weight/weight molar ratio of about 30:70 of cationic surfactant and anionic surfactant in aqueous solution to the bacterial cells; (d) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d).

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL from about 7 to about 8:1 comprising: (a) centrifuging bacterial cells; (b) removing any bacterial growth medium; (c) adding a weight/weight molar ratio of about 70:30 of cationic surfactant and anionic surfactant in aqueous solution to the bacterial cells; (d) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d).

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL from about 0.5 to about 1.5:1 comprising: (a) centrifuging bacterial cells; (b) removing any bacterial growth medium; (c) adding a weight/weight molar ratio of about 30:70 of cationic surfactant and anionic surfactant in aqueous solution to from about 10⁶ to about 10⁹ bacterial cells; (d) stirring resulting mixture at room temperature for at least about 15 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d).

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL from about 0.5 to about 1.5:1 comprising: (a) centrifuging bacterial cells; (b) removing any bacterial growth medium; (c) adding a weight/weight molar ratio of about 70:30 of cationic surfactant and anionic surfactant in aqueous solution to from about 10⁶ to about 10⁹ bacterial cells; (d) stirring resulting mixture at room temperature for at least about 15 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(d).

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL of 1: from about 1 to about 1.5 comprising: (a) suspending from about 10⁶ to about 10⁹ bacterial cells in an aqueous solution comprising a volume/volume ratio of preformed catanionic vesicles of about 1% negatively charged surfactamt and water of about 1 to about 2:1; (b) stirring the aqueous solution for about 1 hour; wherein the method does not comprise a step of purification prior to steps (a)-(b).

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL of 1: from about 1 to about 1.5 comprising: (a) suspending from about 10⁶ to about 10⁹ bacterial cells in an aqueous solution comprising a volume/volume ratio of preformed catanionic vesicles of about 1% positively charged surfactamt and water of about 1 to about 2:1; (b) stirring the aqueous solution for about 1 hour; wherein the method does not comprise a step of purification prior to steps (a)-(b).

Aspects of the invention relate to a method of forming catanionic vesicles with a ratio of protein to carbohydrate in micrograms per mL of 1: from about 1 to about 1.5 comprising: (a) suspending from about 10⁶ to about 10⁹ bacterial cells in an aqueous solution comprising a volume/volume ratio of preformed catanionic vesicles of about 1% SDBS and water of about 1 to about 2:1; (b) stirring the aqueous solution for about 1 hour; wherein the method does not comprise a step of purification prior to steps (a)-(b).

In any of the disclosed methods of making vesicles, the amount of bacterial cells used can be from about 10⁶ to about 10⁹ bacteria per mL of solution. In the case of centrifuged cells, one of ordinary skill in the art can take a density measurement to approximate the number of bacterial cells in a solution and add the appropriate volume with a known density to arrive at the number of cells. In any of the disclosed methods of making vesicles, the amount of bacterial cells used can be about 10¹, about 10², about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, to 10⁸, about 10⁹ bacteria per mL of culture medium. In any of the disclosed methods of making vesicles, the number of bacterial cells for each step can utilize from about 10 to about 10¹⁰⁰ bacterial cells. In some embodiments, the above-mentioned methods do not comprise a step of purification or extraction of LPS or LOS prior to steps (a)-(b).

In some embodiments, the vesicles, compositions comprising vesicles, pharmaceutical compositions comprising vesicles, and in accordance with the present invention may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition, such as tularemia. In some embodiments, inventive targeted particles may be used to treat cancer. In certain embodiments, inventive targeted particles may be used to treat prostate cancer.

Cancer can be associated with a variety of physical symptoms. Symptoms of cancer generally depend on the type and location of the tumor. For example, lung cancer can cause coughing, shortness of breath, and chest pain, while colon cancer often causes diarrhea, constipation, and blood in the stool. However, to give but a few examples, the following symptoms are often generally associated with many cancers: fever, chills, night sweats, cough, dyspnea, weight loss, loss of appetite, anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly, hemoptysis, fatigue, malaise, cognitive dysfunction, depression, hormonal disturbances, neutropenia, pain, non-healing sores, enlarged lymph nodes, peripheral neuropathy, and sexual dysfunction.

In one aspect of the disclosure, a method for the treatment of cancer (e.g. breast cancer) is provided. In some embodiments, the treatment of cancer comprises administering a therapeutically effective amount of inventive targeted particles to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention a “therapeutically effective amount” of a pharmaceutical composition comprising a catanionic vesicle disclosed herein is that amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of cancer.

In one aspect of the invention, a method for administering inventive compositions to a subject suffering from cancer (e.g. prostate cancer) is provided. In some embodiments, such methods comprise administering a therapeutically effective amount of inventive targeted particles to a subject in such amounts and for such time as is necessary to achieve the desired result (i.e. treatment of cancer). In certain embodiments of the present invention a “therapeutically effective amount” of an inventive targeted particle is that amount effective for treating, alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of cancer.

Inventive therapeutic protocols involve administering a therapeutically effective amount of an inventive targeted particle to a healthy individual (i.e. a subject who does not display any symptoms of cancer and/or who has not been diagnosed with cancer). For example, healthy individuals may be “immunized” with an inventive targeted particle prior to development of cancer and/or onset of symptoms of cancer; at risk individuals (e.g., patients who have a family history of cancer; patients carrying one or more genetic mutations associated with development of cancer; patients having a genetic polymorphism associated with development of cancer; patients infected by a virus associated with development of cancer; patients with habits and/or lifestyles associated with development of cancer; etc.) can be treated substantially contemporaneously with (e.g., within 48 hours, within 24 hours, or within 12 hours of) the onset of symptoms of cancer. Of course individuals known to have cancer may receive inventive treatment at any time.

In one aspect of the disclosure, a method of vaccinating a subject is provided. In some embodiments, the subject is in need of treatment or prevention for or from a particular pathogen infection, such as an infection with a Gram-negative bacteria, and the method comprises administering to the subject in need of treatment or prevention a therapeutically effective amount of the vaccine, pharmaceutical composition or composition disclosed herein. In one aspect of the disclosure, a method of stimulating an antigen-specific immune response in a subject is provided. In one aspect of the disclosure, a method of adjuvating an antigen-specific immune response in a subject is provided. by administering any one or plurality of compositions or pharmaceutical compositions disclosed herein in combination with a vaccine or other active agent.

In another aspect of the disclosure, a method of treating and/or preventing tularemia is provided by administering to a subject in need thereof any one or plurality of composition, vaccines or pharmaceutical compositions disclosed herein.

In another aspect of the disclosure, a method of treating and/or preventing gonorrhea is provided by administering to a subject in need thereof any one or plurality of composition, vaccines or pharmaceutical compositions disclosed herein.

In another aspect of the disclosure, a method of treating and/or preventing meningitis is provided by administering to a subject in need thereof any one or plurality of composition, vaccines or pharmaceutical compositions disclosed herein.

In another aspect of the disclosure, a method of treating and/or preventing a bacterial infection of Gram negative or Gram positive bacteria is provided by administering to a subject in need thereof any one or plurality of composition, vaccines or pharmaceutical compositions disclosed herein.

The present disclosure relates to a method of reducing the toxicity of a lipopolysaccharide or a lipooligosaccharide by exposing the lipopolysaccharide or a lipooligosaccharide of a bacterial cell to any of the methods disclosed herein.

In some embodiments, the disclosure relates to a method of manufacturing an antibody against an antigen comprising injecting any of the compositions disclosed herein into a subject and subsequently collecting its serum.

The present disclosure relates to a method of immunizing a subject by administration of any composition or vaccine disclosed herein. The vaccines of the invention can be administered via any route, including but not limited to intramuscular, intraperitoneal, intravenous, intranasally, and the like. Routes of administration include, but are not limited to, intramuscular, intransally, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterially, intraoccularly and oral as well as topically, transdermally, by inhalation or suppository or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue. In some embodiments, the method comprises administering the composition or any of the compositions disclosed herein more than once, twice, or three times to elicit a protective antigen-specific immune response and maintain or boost antibody production. In some embodiments, the method include a dosing regimen with more than one mode of administration comprises. For instance, in the case of two different modes of administration, a method may include intranasal immunization followed by an intraperitoneal immunization or intraperitoneal immunization followed by intanasal immunization.

Kits

The present invention provides kits useful for carrying out various aspects of the invention. In some embodiments, a kit may include, for example, (i) any one or plurality of vesicles disclosed herein, optionally comprising a targeting moiety, and one or more particular therapeutic agents to be delivered; and (ii) instructions for administering the targeted particle to a subject in need thereof. In some embodiments, a kit may include, for example, (i) any one or plurality of vesicles and vaccines disclosed herein in a first container optionally comprising one or more particular therapeutic agents; and a syringe or other device to administer the vesicle or vaccine. In some embodiments, a kit may include instructions for administering the vaccine to a subject in need thereof. In some embodiments, a kit may be provided which includes materials useful for identifying and/or screening for novel targeting moieties. Such a kit may include, for example, (i) any one or plurality of vesicles disclosed herein, a library of targeting vesicles comprising any one or plurality of bacterial antigens, and one or more therapeutic agents to be delivered; (ii) a targeted particle or vesicle disclosed herein that may serve as a positive control; and (iii) a targeted particle or vesicle disclosed herein that may serve as a negative control.

TABLE 1 UniProtA C Desc Peptide SEQ ID NO:  Q5F905 30S ribosomal protein S1 NAQGEIEVK SEQ ID NO: 2 Q5F905 30S ribosomal protein S1 VGDFVTVTIESVENGFGETK SEQ ID NO: 3 Q5F905 30S ribosomal protein S1 GGLTVMISSIR SEQ ID NO: 4 Q5F905 30S ribosomal protein S1 AFLPGSLVDVRPVK SEQ ID NO: 5 Q5F905 30S ribosomal protein S1 AFLPGSLVDVRPVKDTSHFEGK SEQ ID NO: 6 Q5F905 30S ribosomal protein S1 RAVLEATLGEER SEQ ID NO: 7 Q5F905 30S ribosomal protein S1 AVLEATLGEERK SEQ ID NO: 8 Q5F905 30S ribosomal protein S1 KALLENLQEGSVIK SEQ ID NO: 9 Q5F905 30S ribosomal protein S1 ALLENLQEGSVIK SEQ ID NO: 10 Q5F905 30S ribosomal protein S1 RVKHPSEVLEVGQEVEAK SEQ ID NO: 11 Q5F905 30S ribosomal protein S1 VKHPSEVLEVGQEVEAK SEQ ID NO: 12 Q5F905 30S ribosomal protein S1 QLGEDPWSGLTR SEQ ID NO: 13 Q5F905 30S ribosomal protein S1 RYPQATR SEQ ID NO: 14 Q5F905 30S ribosomal protein S1 QLEGDPFGNFISVNDKGSLVK SEQ ID NO: 15 Q5F905 30S ribosomal protein S1 LKEGDEVEAVIVTVDRK SEQ ID NO: 16 Q5F905 30S ribosomal protein S1 EALNSVNAAANANAGTTSLGDLLK SEQ ID NO: 17 Q5F9P8 5′-methylthioadenosine/S- GQVPQLPAR SEQ ID NO: 18 adenosylhomocysteine nucleosidase Q5F9P8 5′-methylthioadenosine/S- FASDGILIETAKR SEQ ID NO: 19 adenosylhomocysteine nucleosidase Q5FA17 ABC transporter, GDQLAGQIKEEGSR SEQ ID NO: 20 periplasmic binding protein, iron related Q5FA17 ABC transporter, LEAPQVSATTVSEKEHATR SEQ ID NO: 21 periplasmic binding protein, iron related Q5F7L8 ABC transporter, VASNAEFAPFESLDSK SEQ ID NO: 22 periplasmic histidine- binding protein Q5F7L8 ABC transporter, GNVEGFDVDLMNAMAK SEQ ID NO: 23 periplasmic histidine- binding protein Q5F7L8 ABC transporter, AGNFKIEFK SEQ ID NO: 24 periplasmic histidine- binding protein Q5F7L8 ABC transporter, QSMDFSDPYFEITQVVLVPK SEQ ID NO: 25 periplasmic histidine- binding protein Q5F7L8 ABC transporter, VGVVTGHTGDFSVSK SEQ ID NO: 26 periplasmic histidine- binding protein Q5F7L8 ABC transporter, LLGNDNPK SEQ ID NO: 27 periplasmic histidine- binding protein Q5F7L8 ABC transporter, FENVPLIIK SEQ ID NO: 28 periplasmic histidine- binding protein Q5F7L8 ABC transporter, ELENGGLDSVVSDSAVIANYVK SEQ ID NO: 29 periplasmic histidine- binding protein Q5F7L8 ABC transporter, GMDFVTLPDFTTEHYGIAVR SEQ ID NO: 30 periplasmic histidine- binding protein Q5F824 Acetate kinase LILVLNCGSSSLK SEQ ID NO: 31 Q5F824 Acetate kinase VVSGGELYNESILVDDEVIAGIEK SEQ ID NO: 32 Q5F824 Acetate kinase CIPLAPLHNPAHLLGLR SEQ ID NO: 33 Q5F824 Acetate kinase GLPNVVVFDTSFHQTMPEVAYK SEQ ID NO: 34 Q5F824 Acetate kinase YGAHGTSYR SEQ ID NO: 35 Q5F824 Acetate kinase FVADETAHFLGK SEQ ID NO: 36 Q5F824 Acetate kinase FVADETAHFLGKDKK SEQ ID NO: 37 Q5F824 Acetate kinase DTSMGLTPLEGLVMGTR SEQ ID NO: 38 Q5F824 Acetate kinase TIEEEAAKGHKGAK SEQ ID NO: 39 Q5F824 Acetate kinase FGNAGVITTADSK SEQ ID NO: 40 Q5F824 Acetate kinase AVAVVIPTNEELMIAHDTAR SEQ ID NO: 41 Q5F9Y5 Acetyl-coenzyme A CNHHNPLSAR SEQ ID NO: 42 carboxylase carboxyl transferase subunit beta Q5F9Y5 Acetyl-coenzyme A FIGGSMGSVVGER SEQ ID NO: 43 carboxylase carboxyl transferase subunit beta Q5F9Y5 Acetyl-coenzyme A TSAALHLLTEK SEQ ID NO: 44 carboxylase carboxyl transferase subunit beta Q5F7E7 Aconitate hydratase 2 AALGIPALPLNAQQTADLVELLK SEQ ID NO: 45 Q5F7E7 Aconitate hydratase 2 NPPAGEGEFLVELLAHR SEQ ID NO: 46 Q5F7E7 Aconitate hydratase 2 VKASFLAAVAEGSASSPLVSPK SEQ ID NO: 47 Q5F7E7 Aconitate hydratase 2 ASFLAAVAEGSASSPLVSPK SEQ ID NO: 48 Q5F7E7 Aconitate hydratase 2 AKVPEKITVTVFK SEQ ID NO: 49 Q5F7E7 Aconitate hydratase 2 DGITPDKPGEVGPIK SEQ ID NO: 50 Q5F7E7 Aconitate hydratase 2 DGITPDKPGEVGPIKLLEELK SEQ ID NO: 51 Q5F7E7 Aconitate hydratase 2 AKGHPVAYVGDVVGTGSSR SEQ ID NO: 52 Q5F7E7 Aconitate hydratase 2 GHPVAYVGDVVGTGSSR SEQ ID NO: 53 Q5F7E7 Aconitate hydratase 2 GHPVAYVGDVVGTGSSRK SEQ ID NO: 54 Q5F7E7 Aconitate hydratase 2 KSATNSVIWHTGEDIPFVPNKR SEQ ID NO: 55 Q5F7E7 Aconitate hydratase 2 SATNSVIWHTGEDIPFVPNKR SEQ ID NO: 56 Q5F7E7 Aconitate hydratase 2 IAPIFFNTQEDSGALPIEVDVSALK SEQ ID NO: 57 Q5F7E7 Aconitate hydratase 2 SQVLLDEVQAGGR SEQ ID NO: 58 Q5F7E7 Aconitate hydratase 2 ACGLPEGQGVRPGTYCEPR SEQ ID NO: 59 Q5F7E7 Aconitate hydratase 2 MTTVGSQDTTGPMTR SEQ ID NO: 60 Q5F7E7 Aconitate hydratase 2 THKELPAFISTR SEQ ID NO: 61 Q5F7E7 Aconitate hydratase 2 GGVSLRPGDGVIHSWLNR SEQ ID NO: 62 Q5F7E7 Aconitate hydratase 2 DLVNAIPLYAIK SEQ ID NO: 63 Q5F7E7 Aconitate hydratase 2 LNKEPIIEYMK SEQ ID NO: 64 Q5F7E7 Aconitate hydratase 2 LLEGKSDIPVR SEQ ID NO: 65 Q5F7E7 Aconitate hydratase 2 ELSDEGHYGVLGR SEQ ID NO: 66 Q5F7E7 Aconitate hydratase 2 EGATVMSTSTR SEQ ID NO: 67 Q5F7E7 Aconitate hydratase 2 NTFVYLGSAELAAICSK SEQ ID NO: 68 Q5F7E7 Aconitate hydratase 2 LGKIPTVEEYQANIGIINEQGDKIYR SEQ ID NO: 69 Q5F7E7 Aconitate hydratase 2 IPTVEEYQANIGIINEQGDKIYR SEQ ID NO: 70 Q5F7T9 Alpha-2,3- NASDEHYTIFK SEQ ID NO: 71 sialyltransferase Q5F7T9 Alpha-2,3- KMTYLPLFDASELKAGDETGGTVR SEQ ID NO: 72 sialyltransferase Q5F7T9 Alpha-2,3- ILLGSPDKEMKEISEK SEQ ID NO: 73 sialyltransferase Q5F7T9 Alpha-2,3- NFNIQYVAPHPR SEQ ID NO: 74 sialyltransferase Q5F6Y6 Aminomethyltransferase AFFRKLIANDVAK SEQ ID NO: 75 Q5F6Y6 Aminomethyltransferase ALQTAGVQPCGLGAR SEQ ID NO: 76 Q5F724 Antibiotic resistance AQVGGIIQK SEQ ID NO: 77 efflux pump component Q5F724 Antibiotic resistance AQVGGIIQKR SEQ ID NO: 78 efflux pump component Q5F724 Antibiotic resistance AGQPLYQIDSSTYEAGLESAR SEQ ID NO: 79 efflux pump component Q5F724 Antibiotic resistance AQLATAQATLAK SEQ ID NO: 80 efflux pump component Q5F724 Antibiotic resistance SAEAGVKAAQAAIK SEQ ID NO: 81 efflux pump component Q5F724 Antibiotic resistance SAGINLNR SEQ ID NO: 82 efflux pump component Q5F724 Antibiotic resistance SRITAPISGFIGQSK SEQ ID NO: 83 efflux pump component Q5F724 Antibiotic resistance ITAPISGFIGQSK SEQ ID NO: 84 efflux pump component Q5F724 Antibiotic resistance VSEGTLLNAGDTTVLATIR SEQ ID NO: 85 efflux pump component Q5F724 Antibiotic resistance QTNPMYVNVTQSASEVMK SEQ ID NO: 86 efflux pump component Q5F724 Antibiotic resistance AAVSNDQNILMPGLYVR SEQ ID NO: 87 efflux pump component Q5F724 Antibiotic resistance VLMDQVAADNAFIVPQQAVTR SEQ ID NO: 88 efflux pump component Q5F724 Antibiotic resistance GAKDTVMIVNAQGGMEPR SEQ ID NO: 89 efflux pump component Q5F724 Antibiotic resistance EWAPSENQAAAPQAGVQTASEAKPASEAK SEQ ID NO: 90 efflux pump component Q5F725 Antibiotic resistance LSEVLSTLPATVQQYGVTVSK SEQ ID NO: 91 efflux pump component Q5F725 Antibiotic resistance ANTDGSNIYLKDVAK SEQ ID NO: 92 efflux pump component Q5F725 Antibiotic resistance TDATLAQVTQLAK SEQ ID NO: 93 efflux pump component Q5F725 Antibiotic resistance TASGSDAVAVAGK SEQ ID NO: 94 efflux pump component Q5F725 Antibiotic resistance ASGLFDPSTVR SEQ ID NO: 95 efflux pump component Q5F725 Antibiotic resistance AGGLEDSPQLK SEQ ID NO: 96 efflux pump component Q5F725 Antibiotic resistance AAAAAQGISFADIR SEQ ID NO: 97 efflux pump component Q5F725 Antibiotic resistance TALASALSSSYVSDFPNQGR SEQ ID NO: 98 efflux pump component Q5F725 Antibiotic resistance MQPADILNLTVPNK SEQ ID NO: 99 efflux pump component Q5F725 Antibiotic resistance SGVAVPLSTIATVSWENGTEQSVR SEQ ID NO: 100 efflux pump component Q5F725 Antibiotic resistance LSASPATGVSTGQAMAAVQK SEQ ID NO: 101 efflux pump component Q5F725 Antibiotic resistance AGITGSDDKQY SEQ ID NO: 102 efflux pump component Q5F842 Aspartokinase YGGTSVGSPER SEQ ID NO: 103 Q5F842 Aspartokinase AAVTGIAFDKNQAR SEQ ID NO: 104 Q5FAH2 Carbamoyl-phosphate TPASFEPSIDYVVTK SEQ ID NO: 105 synthase large chain Q5FAH2 Carbamoyl-phosphate ELANPGPER SEQ ID NO: 106 synthase large chain Q5FAH2 Carbamoyl-phosphate VLNDLGLRQPPNR SEQ ID NO: 107 synthase large chain Q5FAH2 Carbamoyl-phosphate IAHNEEEALVK SEQ ID NO: 108 synthase large chain Q5FAH2 Carbamoyl-phosphate AEEIGYPLVVRPSYVLGGR SEQ ID NO: 109 synthase large chain Q5FAH2 Carbamoyl-phosphate VPQYTTTAGGEAMSEGAK SEQ ID NO: 110 synthase large chain Q5F601 Catalase TTSKCPVTHLTMNNGAPVADNQNSLTAGPR SEQ ID NO: 111 Q5F601 Catalase CPVTHLTMNNGAPVADNQNSLTAGPR SEQ ID NO: 112 Q5F601 Catalase GPLLTQDLWLNEK SEQ ID NO: 113 Q5F601 Catalase EVIPERR SEQ ID NO: 114 Q5F601 Catalase FTTVAGER SEQ ID NO: 115 Q5F601 Catalase LFNYADAQR SEQ ID NO: 116 Q5F601 Catalase QIPVNRPR SEQ ID NO: 117 Q5F601 Catalase ALFNLMNDAQK SEQ ID NO: 118 Q5F601 Catalase QALFDNTAAAMGDAPDFIK SEQ ID NO: 119 Q5F601 Catalase QALFDNTAAAMGDAPDFIKYR SEQ ID NO: 120 Q5F601 Catalase CDPAYGEGVAK SEQ ID NO: 121 Q5F6M2 Cell division protein ftsA ITHVTTGIAGNHIR SEQ ID NO: 122 Q5F6M2 Cell division protein ftsA VHIITGASTAVQNVQK SEQ ID NO: 123 Q5F6M3 Cell division protein FtsZ MEFVYDVAESAVSPAVIK SEQ ID NO: 124 Q5F6M3 Cell division protein FtsZ VIGLGGGGCNAINNMVANNVR SEQ ID NO: 125 Q5F6M3 Cell division protein FtsZ SVEFISANTDAQSLAK SEQ ID NO: 126 Q5F6M3 Cell division protein FtsZ RIQLGTNLTR SEQ ID NO: 127 Q5F6M3 Cell division protein FtsZ IQLGTNLTR SEQ ID NO: 128 Q5F6M3 Cell division protein FtsZ GLGAGANPDIGR SEQ ID NO: 129 Q5F6M3 Cell division protein FtsZ GIAMMGSGYAQGIDR SEQ ID NO: 130 Q5F6M3 Cell division protein FtsZ MATDQAISSPLLDDVTLDGAR SEQ ID NO: 131 Q5F6M3 Cell division protein FtsZ GVLVNITTAPGCLK SEQ ID NO: 132 Q5F6M3 Cell division protein FtsZ IVNQSAHPDLECK SEQ ID NO: 133 Q5F6M3 Cell division protein FtsZ ITIIATGLKEK SEQ ID NO: 134 Q5F6M3 Cell division protein FtsZ GAVDPTPAR SEQ ID NO: 135 Q5F6M3 Cell division protein FtsZ GAVDPTPAREVEAVAPSKQEQSHNVEGMIR SEQ ID NO: 136 Q5F6M3 Cell division protein FtsZ EVEAVAPSKQEQSHNVEGMIR SEQ ID NO: 137 Q5F9Z8 Cell division protein ZipA VRDQFGHSDKDALLNSK SEQ ID NO: 138 Q5F9Z8 Cell division protein ZipA TSHVRDGKPSGGPVMMPKPQPAVK SEQ ID NO: 139 Q5F9Z8 Cell division protein ZipA KPAKPQDSAMR SEQ ID NO: 140 Q5F9Z8 Cell division protein ZipA NLQEQDAVYIAK SEQ ID NO: 141 Q5F873 Citrate synthase SKSIKLNVPGR SEQ ID NO: 142 Q5F873 Citrate synthase SIKLNVPGR SEQ ID NO: 143 Q5F873 Citrate synthase AGLELPVLEASIGHDVVDIR SEQ ID NO: 144 Q5F873 Citrate synthase IFILHADHEQNASTSTVR SEQ ID NO: 145 Q5F873 Citrate synthase RDYVPAGER SEQ ID NO: 146 Q5F9Q2 Cysteine synthase MKIANSITELIGNTPLVK SEQ ID NO: 147 Q5F9Q2 Cysteine synthase IANSITELIGNTPLVK SEQ ID NO: 148 Q5F9Q2 Cysteine synthase GLKAEVAVK SEQ ID NO: 149 Q5F9Q2 Cysteine synthase TFGAELILTPAAEGMAGAIAK SEQ ID NO: 150 Q5F9Q2 Cysteine synthase AQSLVDAHPDTYFMPR SEQ ID NO: 151 Q5F9Q2 Cysteine synthase QFDNEANPEVHR SEQ ID NO: 152 Q5F9Q2 Cysteine synthase QFDNEANPEVHRK SEQ ID NO: 153 Q5F9Q2 Cysteine synthase GPHPIQGIGAGFIPTVLNTK SEQ ID NO: 154 Q5F9Q2 Cysteine synthase IYDSIAKVPNEAAFETAR SEQ ID NO: 155 Q5F9Q2 Cysteine synthase VPNEAAFETAR SEQ ID NO: 156 Q5F9Q2 Cysteine synthase EGILAGISSGAAVWSALQLAK SEQ ID NO: 157 Q5F9Q2 Cysteine synthase QPENEGKLIVVLLPSYGER SEQ ID NO: 158 Q5F9Q2 Cysteine synthase LIVVLLPSYGER SEQ ID NO: 159 Q5F9K8 Delta-aminolevulinic acid LGIPMLALFPVVTANK SEQ ID NO: 160 dehydratase Q5F9K8 Delta-aminolevulinic acid AQEAYNPEGLVPSTVR SEQ ID NO: 161 dehydratase Q5F876 Dihydrolipoyl SQYDVVVIGAGPGGYVAAIR SEQ ID NO: 162 dehydrogenase Q5F876 Dihydrolipoyl TNLPNVWAIGDVVR SEQ ID NO: 163 dehydrogenase Q5F876 Dihydrolipoyl ASDEGVAVAER SEQ ID NO: 164 dehydrogenase Q5F8Y0 DNA gyrase subunit AIVGDVIGK SEQ ID NO: 165 Q5F8Y0 DNA gyrase subunit AIIDFVDILSKPER SEQ ID NO: 166 Q5F8Y0 DNA gyrase subunit AKNKGGQGSIAINTGER SEQ ID NO: 167 Q5F8Y0 DNA gyrase subunit ALINLDEGETLVSLER SEQ ID NO: 168 Q5F5M0 DNA-binding competence VKGIGPAVLAK SEQ ID NO: 169 protein 2 Q5F5M0 DNA-binding competence LKDQASVGAPAPK SEQ ID NO: 170 protein 2 Q5F696 Enoyl-[acyl-carrier- EAFNTAHEISAYSLPALAK SEQ ID NO: 171 protein] reductase [NADH] Q5F696 Enoyl-[acyl-carrier- TLAASGIADFGK SEQ ID NO: 172 protein] reductase [NADH] Q5F8I5 Genome-derived Neisseria AMQAQITAER SEQ ID NO: 173 antigen 1220 Q5F8I5 Genome-derived Neisseria AMQAQITAEREK SEQ ID NO: 174 antigen 1220 Q5F8I5 Genome-derived Neisseria IAESEGRKIEQINLASGQR SEQ ID NO: 175 antigen 1220 Q5F8I5 Genome-derived Neisseria KIEQINLASGQR SEQ ID NO: 176 antigen 1220 Q5F8I5 Genome-derived Neisseria IEQINLASGQR SEQ ID NO: 177 antigen 1220 Q5F8I5 Genome-derived Neisseria EAEIQQSEGEAQAAVNASNAEK SEQ ID NO: 178 antigen 1220 Q5F8I5 Genome-derived Neisseria QIAAALQTQGGADAVNLK SEQ ID NO: 179 antigen 1220 Q5F8I5 Genome-derived Neisseria IAEQYVAAFNNLAK SEQ ID NO: 180 antigen 1220 Q5F731 Glutamate dehydrogenase TDLNTLFANLK SEQ ID NO: 181 Q5F731 Glutamate dehydrogenase TDLNTLFANLKQR SEQ ID NO: 182 Q5F731 Glutamate dehydrogenase NPKYTQQSLLER SEQ ID NO: 183 Q5F731 Glutamate dehydrogenase YTQQSLLER SEQ ID NO: 184 Q5F731 Glutamate dehydrogenase FLAFEQVFK SEQ ID NO: 185 Q5F731 Glutamate dehydrogenase VLISGSGNVAQYAAEK SEQ ID NO: 186 Q5F731 Glutamate dehydrogenase ASNAGGVATSGLEMSQNAIR SEQ ID NO: 187 Q5F731 Glutamate dehydrogenase VGDKVNYVNGANIAGFVK SEQ ID NO: 188 Q5F6F9 Glutamine synthetase GGYAPVAPIDCGQDLR SEQ ID NO: 189 Q5F6F9 Glutamine synthetase FATLVKR SEQ ID NO: 190 Q5F6F9 Glutamine synthetase ALNAITNPSTNSYK SEQ ID NO: 191 Q5F6F9 Glutamine synthetase ALNAITNPSTNSYKR SEQ ID NO: 192 Q5F6F9 Glutamine synthetase LVPHFEAPTK SEQ ID NO: 193 Q5F6F9 Glutamine synthetase LVPHFEAPTKLAYSAK SEQ ID NO: 194 Q5F6F9 Glutamine synthetase SASIRIPSVNSSK SEQ ID NO: 195 Q5F7G1 Glutathione synthetase AADKVQTALK SEQ ID NO: 196 Q5F7G1 Glutathione synthetase FTAPTLVTTR SEQ ID NO: 197 Q5F7G1 Glutathione synthetase YIPEIVHGDKR SEQ ID NO: 198 Q5F7G1 Glutathione synthetase ILIIGGEVVPYALAR SEQ ID NO: 199 Q5F8H4 Inosine-5′-monophosphate AYTFDDVLLVPAHSTVLPR SEQ ID NO: 200 dehydrogenase Q5F8H4 Inosine-5′-monophosphate EITLNLPLLSAAMDTVTEAR SEQ ID NO: 201 dehydrogenase Q5F8H4 Inosine-5′-monophosphate RHESGVVKDPVTVAPTTLIR SEQ ID NO: 202 dehydrogenase Q5F8H4 Inosine-5′-monophosphate HESGVVKDPVTVAPTTLIR SEQ ID NO: 203 dehydrogenase Q5F8H4 Inosine-5′-monophosphate TTEFPNANKDSEGR SEQ ID NO: 204 dehydrogenase Q5F8H4 Inosine-5′-monophosphate VGAAVGTGGDTDERVK SEQ ID NO: 205 dehydrogenase Q5F8H4 Inosine-5′-monophosphate IVAGVGVPQLTAIHNVAEALK SEQ ID NO: 206 dehydrogenase Q5F809 Lipid modified azurin ACKEFTITLK SEQ ID NO: 207 protein*** Q5F809 Lipid modified azurin ASMGHNLVIAK SEQ ID NO: 208 protein Q5F809 Lipid modified azurin LIGGGEESSLTLDPAK SEQ ID NO: 209 protein Q5F809 Lipid modified azurin FACTFPGHGALMNGK SEQ ID NO: 210 protein Q5F809 Lipid modified azurin FACTFPGHGALMNGKVTLVD SEQ ID NO: 211 protein Q5F501 Lipoprotein*** DSAPAASAAAPSADNGAAK SEQ ID NO: 212 Q5F501 Lipoprotein DSAPAASAAAPSADNGAAKK SEQ ID NO: 213 Q5F501 Lipoprotein EIVFGTTVGDFGDMVK SEQ ID NO: 214 Q5F501 Lipoprotein EQIQAELEKK SEQ ID NO: 215 Q5F501 Lipoprotein EHNLDITEAFQVPTAPLGLYPGK SEQ ID NO: 216 Q5F501 Lipoprotein LKSLEEVKDGSTVSAPNDPSNFAR SEQ ID NO: 217 Q5F501 Lipoprotein SLEEVKDGSTVSAPNDPSNFAR SEQ ID NO: 218 Q5F501 Lipoprotein DGSTVSAPNDPSNFAR SEQ ID NO: 219 Q5F501 Lipoprotein ALVMLNELGWIK SEQ ID NO: 220 Q5F501 Lipoprotein LKDGINPLTASK SEQ ID NO: 221 Q5F501 Lipoprotein IVELEAAQLPR SEQ ID NO: 222 Q5F501 Lipoprotein LTEALFQEPSFAYVNWSAVK SEQ ID NO: 223 Q5F501 Lipoprotein TADKDSQWLKDVTEAYNSDAFK SEQ ID NO: 224 Q5F501 Lipoprotein RFEGYKYPAAWNEGAAK SEQ ID NO: 225 Q5F501 Lipoprotein FEGYKYPAAWNEGAAK SEQ ID NO: 226 Q5F5V7 Major outer membrane AGVQTYRSVEHTDGK SEQ ID NO: 227 protein porin P.IB Q5F5V7 Major outer membrane AGVQTYRSVEHTDGKVSK SEQ ID NO: 228 protein porin P.IB Q5F5V7 Major outer membrane AGVQTYRSVEHTDGKVSKVETGSEIADFGSK SEQ ID NO: 229 protein porin P.IB Q5F5V7 Major outer membrane SVEHTDGKVSK SEQ ID NO: 230 protein porin P.IB Q5F5V7 Major outer membrane SVEHTDGKVSKVETGSEIADFGSK SEQ ID NO: 231 protein porin P.IB Q5F5V7 Major outer membrane VSKVETGSEIADFGSK SEQ ID NO: 232 protein porin P.IB Q5F5V7 Major outer membrane VETGSEIADFGSK SEQ ID NO: 233 protein porin P.IB Q5F5V7 Major outer membrane IGFKGQEDLGNGLK SEQ ID NO: 234 protein porin P.IB Q5F5V7 Major outer membrane GQEDLGNGLK SEQ ID NO: 235 protein porin P.IB Q5F5V7 Major outer membrane AVWQLEQGASVAGTNTGWGNK SEQ ID NO: 236 protein porin P.IB Q5F5V7 Major outer membrane AVWQLEQGASVAGTNTGWGNKQSFVGLK SEQ ID NO: 237 protein porin P.IB Q5F5V7 Major outer membrane QSFVGLKGGFGTIR SEQ ID NO: 238 protein porin P.IB Q5F5V7 Major outer membrane AGSLNSPLK SEQ ID NO: 239 protein porin P.IB Q5F5V7 Major outer membrane FTGNVLEISGMAQR SEQ ID NO: 240 protein porin P.IB Q5F5V7 Major outer membrane YDSPEFAGFSGSVQYAPK SEQ ID NO: 241 protein porin P.IB Q5F5V7 Major outer membrane LVGGYDNNALYVSVAAQQQDAK SEQ ID NO: 242 protein porin P.IB Q5F5V7 Major outer membrane VSYAHGFK SEQ ID NO: 243 protein porin P.IB Q5F5V7 Major outer membrane GTVDSANHDNTYDQVVVGAEYDFSKR SEQ ID NO: 244 protein porin P.IB Q5F5V7 Major outer membrane RTSALVSAGWLQEGK SEQ ID NO: 245 protein porin P.IB Q5F5V7 Major outer membrane TSALVSAGWLQEGK SEQ ID NO: 246 protein porin P.IB Q5F5V7 Major outer membrane TSALVSAGWLQEGKGADK SEQ ID NO: 247 protein porin P.IB Q5F5V7 Major outer membrane TSALVSAGWLQEGKGADKIVSTASAVVLR SEQ ID NO: 248 protein porin P.IB Q5F5V7 Major outer membrane GADKIVSTASAVVLR SEQ ID NO: 249 protein porin P.IB Q5F5V7 Major outer membrane IVSTASAVVLR SEQ ID NO: 250 protein porin P.IB Q5F4X7 Malonyl CoA-acyl carrier TVEIKQPQIR SEQ ID NO: 251 protein transacylase Q5F4X7 Malonyl CoA-acyl carrier VIHNADVAAYDDAGK SEQ ID NO: 252 protein transacylase Q5F4X7 Malonyl CoA-acyl carrier IKDALVR SEQ ID NO: 253 protein transacylase Q5F726 Multidrug efflux pump NNLLPTLAANANGSR SEQ ID NO: 254 channel protein Q5F726 Multidrug efflux pump AGVISAVALRQQEALIESAK SEQ ID NO: 255 channel protein Q5F726 Multidrug efflux pump NALATLINRPIPEDLPAGLPLDK SEQ ID NO: 256 channel protein Q5F726 Multidrug efflux pump LPAGLSSEVLLDRPDIR SEQ ID NO: 257 channel protein Q5F726 Multidrug efflux pump AAEHALKQANANIGAAR SEQ ID NO: 258 channel protein Q5F726 Multidrug efflux pump QANANIGAAR SEQ ID NO: 259 channel protein Q5F726 Multidrug efflux pump LTGSVGTGSVELGGLFK SEQ ID NO: 260 channel protein Q5F726 Multidrug efflux pump YKHGVSGALDLLDAER SEQ ID NO: 261 channel protein Q5F6N6 Outer membrane opacity NKISTVSDYFR SEQ ID NO: 262 protein D Q5F6N6 Outer membrane opacity ISTVSDYFR SEQ ID NO: 263 protein D Q5F6N6 Outer membrane opacity VSVGYDFGGWR SEQ ID NO: 264 protein D Q5F6N6 Outer membrane opacity VAYGHVR SEQ ID NO: 265 protein D Q5F5W8 Outer membrane protein TEPSTVFNYLPVK SEQ ID NO: 266 assembly factor BamA Q5F5W8 Outer membrane protein VGDTYNDTHGSAIIK SEQ ID NO: 267 assembly factor BamA Q5F5W8 Outer membrane protein GKLNIQITPK SEQ ID NO: 268 assembly factor BamA Q5F5W8 Outer membrane protein ILDTDIQTNEDKTR SEQ ID NO: 269 assembly factor BamA Q5F5W8 Outer membrane protein QQMTAVLGEIQNR SEQ ID NO: 270 assembly factor BamA Q5F5W8 Outer membrane protein IYVNEIHITGNNK SEQ ID NO: 271 assembly factor BamA Q5F5W8 Outer membrane protein TKEIPFFENFYGGGLGSVR SEQ ID NO: 272 assembly factor BamA Q5F9W0 Outer membrane protein GLVLFNEDQSFLNK SEQ ID NO: 273 assembly factor BamD Q5F9W0 Outer membrane protein GAYIAAANR SEQ ID NO: 274 assembly factor BamD Q5F5Y8 Outer membrane protein AVAALRPGMTKDQVLLLLGSPILR SEQ ID NO: 275 assembly factor BamE Q5F6I1 Outer membrane protein NAYFDKASQGR SEQ ID NO: 276 PIII*** Q5F6I1 Outer membrane protein PIII ASQGRVECGDAVAVPEPEPAPVAVVEQAPQYVDETISLSAK SEQ ID NO: 277 Q5F6I1 Outer membrane protein PIII TLFGFDKDSLR SEQ ID NO: 278 Q5F6I1 Outer membrane protein PIII TLFGFDKDSLRAEAQDNLK SEQ ID NO: 279 Q5F6I1 Outer membrane protein PIII DSLRAEAQDNLK SEQ ID NO: 280 Q5F6I1 Outer membrane protein PIII TNVQSVR SEQ ID NO: 281 Q5F6I1 Outer membrane protein PIII VEGHTDFMGSEK SEQ ID NO: 282 Q5F6I1 Outer membrane protein PIII VEGHTDFMGSEKYNQALSER SEQ ID NO: 283 Q5F6I1 Outer membrane protein PIII YNQALSER SEQ ID NO: 284 Q5F6I1 Outer membrane protein PIII RAYVVANNLVSNGVPASR SEQ ID NO: 285 Q5F6I1 Outer membrane protein PIII AYVVANNLVSNGVPASR SEQ ID NO: 286 Q5F6I1 Outer membrane protein PIII ISAVGLGESQAQMTQVCQAEVAK SEQ ID NO: 287 Q5F6I1 Outer membrane protein PIII EALIACIEPDR SEQ ID NO: 288 Q5F6I1 Outer membrane protein PIII EALIACIEPDRRVDVK SEQ ID NO: 289 Q5F6I1 Outer membrane protein PIII IRSIVTR SEQ ID NO: 290 Q5F7F3 Peptidyl-prolyl cis-trans SLKQMKEQGAEIDLK SEQ ID NO: 291 isomerase Q5F7F3 Peptidyl-prolyl cis-trans FLQEQQAK SEQ ID NO: 292 isomerase Q5F7F3 Peptidyl-prolyl cis-trans ANKEKGEAFLKENAAK SEQ ID NO: 293 isomerase Q5F7F3 Peptidyl-prolyl cis-trans ANKEKGEAFLKENAAKDGVK SEQ ID NO: 294 isomerase Q5F7F3 Peptidyl-prolyl cis-trans DGVKTTASGLQYK SEQ ID NO: 295 isomerase Q5F7F3 Peptidyl-prolyl cis-trans DGVKTTASGLQYKITK SEQ ID NO: 296 isomerase Q5F7F3 Peptidyl-prolyl cis-trans TTASGLQYK SEQ ID NO: 297 isomerase Q5F7F3 Peptidyl-prolyl cis-trans TTASGLQYKITK SEQ ID NO: 298 isomerase Q5F7F3 Peptidyl-prolyl cis-trans QGEGKQPTKDDIVTVEYEGR SEQ ID NO: 299 isomerase Q5F7F3 Peptidyl-prolyl cis-trans LIDGTVFDSSK SEQ ID NO: 300 isomerase Q5F7F3 Peptidyl-prolyl cis-trans ANGGPATFPLSQVIPGWTEGVR SEQ ID NO: 301 isomerase Q5F7F3 Peptidyl-prolyl cis-trans LLKEGGEATFYIPSNLAYREQGAGEK SEQ ID NO: 302 isomerase Q5F7F3 Peptidyl-prolyl cis-trans EGGEATFYIPSNLAYR SEQ ID NO: 303 isomerase Q5F7F3 Peptidyl-prolyl cis-trans IGPNATLVFDVK SEQ ID NO: 304 isomerase Q5F7F3 Peptidyl-prolyl cis-trans IGAPENAPAKQPDQVDIK SEQ ID NO: 305 isomerase Q5F7F3 Peptidyl-prolyl cis-trans IGAPENAPAKQPDQVDIKK SEQ ID NO: 306 isomerase Q5F7F3 Peptidyl-prolyl cis-trans IGAPENAPAKQPDQVDIKKVN SEQ ID NO: 307 isomerase Q5F820 Peptidyl-prolyl cis-trans FKATVESVR SEQ ID NO: 308 isomerase Q5F9L7 Peptidyl-prolyl cis-trans TVVQDWGYAVFGK SEQ ID NO: 309 isomerase Q5F9L7 Peptidyl-prolyl cis-trans VVDGFDVVDAIESVSTKR SEQ ID NO: 310 isomerase Q5F668 Phospho-2-dehydro-3- ELLPPIAHLYELPISK SEQ ID NO: 311 deoxyheptonate aldolase Q5F668 Phospho-2-dehydro-3- EASGLVHR SEQ ID NO: 312 deoxyheptonate aldolase Q5FA34 Phosphoenolpyruvate ADNYVIWFENLR SEQ ID NO: 313 synthase Q5FA34 Phosphoenolpyruvate NASLGEMISQLTEK SEQ ID NO: 314 synthase Q5FA34 Phosphoenolpyruvate VPGGFATTADAYR SEQ ID NO: 315 synthase Q5FA34 Phosphoenolpyruvate AFLAHNGLNER SEQ ID NO: 316 synthase Q5FA34 Phosphoenolpyruvate VHKGFEHDIVALSAGVQR SEQ ID NO: 317 synthase Q5FA34 Phosphoenolpyruvate GFEHDIVALSAGVQR SEQ ID NO: 318 synthase Q5FA34 Phosphoenolpyruvate MIFTDKAEAGK SEQ ID NO: 319 synthase Q5FA34 Phosphoenolpyruvate DGLDGKLYILQARPETVK SEQ ID NO: 320 synthase Q5FA34 Phosphoenolpyruvate LYILQARPETVK SEQ ID NO: 321 synthase Q5FA34 Phosphoenolpyruvate VRDEMGLTNVEIMIPFVR SEQ ID NO: 322 synthase Q5FA34 Phosphoenolpyruvate ALKENGLER SEQ ID NO: 323 synthase Q5FAD1 Pilin assembly protein KAELLLNSSDKNTEQAAAPAAEQN SEQ ID NO: 324 Q5F693 Pilus assembly protein KITQEDITVFTR SEQ ID NO: 325 Q5F693 Pilus assembly protein AGLPLMQAFEIVAR SEQ ID NO: 326 Q5F693 Pilus assembly protein GQVEQGSSLSR SEQ ID NO: 327 Q5FADO Pilus assembly protein SIDAASLNNLRDELASIR SEQ ID NO: 328 Q5FADO Pilus assembly protein IAQSPENGGNPDGK SEQ ID NO: 329 Q5FADO Pilus assembly protein SSILNLSAIATTYQAK SEQ ID NO: 330 Q5FAG7 Pilus-associated protein VQMYSASVSTYPGSSSSR SEQ ID NO: 331 Q5FAG7 Pilus-associated protein KDIEGNDSDLAK SEQ ID NO: 332 Q5FAG7 Pilus-associated protein TQNGKYAAFLASGYAAK SEQ ID NO: 333 Q5FAG7 Pilus-associated protein YAAFLASGYAAK SEQ ID NO: 334 Q5FAG7 Pilus-associated protein TIFEGDKPITSAPAVSR SEQ ID NO: 335 Q5FAG7 Pilus-associated protein TVCPNGYVYDKPVNVR SEQ ID NO: 336 Q5F7F1 Probable cytosol FSQAVFHSAHEAAVK SEQ ID NO: 337 aminopeptidase Q5F7F1 Probable cytosol VAEAQVYGQSLCR SEQ ID NO: 338 aminopeptidase Q5F7F1 Probable cytosol TAKAEAEKLGAHAK SEQ ID NO: 339 aminopeptidase Q5F7F1 Probable cytosol GATGRPVPLLMNYLR SEQ ID NO: 340 aminopeptidase Q5FA43 Protein translocase QNITTLHNR SEQ ID NO: 341 subunit SecD Q5F8V3 Pseudouridine synthase QWRDGAAPSAKK SEQ ID NO: 342 Q5F8V3 Pseudouridine synthase GQFYELNPAEVANILK SEQ ID NO: 343 Q5F874 Putative 2-oxoglutarate IASAVAGGADEAMLKK SEQ ID NO: 344 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate IQGVGAAQLDPLK SEQ ID NO: 345 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate dehydrogenase, E1 component IQGVGAAQLDPLKR SEQ ID NO: 346 Q5F874 Putative 2-oxoglutarate dehydrogenase, E1 component NYFESVLSTPHYNADQK SEQ ID NO: 347 Q5F874 Putative 2-oxoglutarate dehydrogenase, E1 component ILKEMTAAETLER SEQ ID NO: 348 Q5F874 Putative 2-oxoglutarate STVHCTDIAK SEQ ID NO: 349 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate EKIETGLPAADIER SEQ ID NO: 350 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate LTEKFTAVPEGFALHPTAK SEQ ID NO: 351 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate FTAVPEGFALHPTAK SEQ ID NO: 352 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate QAIDWGMAETLAYASLLTK SEQ ID NO: 353 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate VVLCAGQVYYDLEAGR SEQ ID NO: 354 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate VEQLYPFPYDEVKAELAK SEQ ID NO: 355 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate NQGAFYQIR SEQ ID NO: 356 dehydrogenase, E1 component Q5F874 Putative 2-oxoglutarate HRIEDVISEEQK SEQ ID NO: 357 dehydrogenase, E1 component Q5F4Y0 Putative 3-oxoacyl-[acyl- IALVTGASR SEQ ID NO: 358 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- GIGAAIADTLAAAGAK SEQ ID NO: 359 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- IIGTATGESGAAAISK SEQ ID NO: 360 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- IIGTATGESGAAAISKR SEQ ID NO: 361 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- LAQWGGEGR SEQ ID NO: 362 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- VLNSAEPETVENLIADIEK SEQ ID NO: 363 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- TFGKLDILVNNAGITR SEQ ID NO: 364 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- LDILVNNAGITR SEQ ID NO: 365 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- ALPEETRQTFTAQTALGR SEQ ID NO: 366 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- FGDAQDIADAVLFLASDQAK SEQ ID NO: 367 carrier protein] reductase Q5F4Y0 Putative 3-oxoacyl-[acyl- YITGQTLHVNGGMLMP SEQ ID NO: 368 carrier protein] reductase Q5F6I5 Putative ABC transporter, LGNEVIEFVNVSK SEQ ID NO: 369 ATP-binding protein Q5F6I5 Putative ABC transporter, VPAGAIVGIIGPNGAGK SEQ ID NO: 370 ATP-binding protein Q5F6I5 Putative ABC transporter, DILQVGQFEIPAR SEQ ID NO: 371 ATP-binding protein Q5F9W7 Putative ABC transporter, KGEVVVVCGPSGSGK SEQ ID NO: 372 ATP-binding protein, amino acid Q5F9W7 Putative ABC transporter, DAFPSQLSGGQQQR SEQ ID NO: 373 ATP-binding protein, amino acid Q5F9W7 Putative ABC transporter, AKQFLQQVMTH SEQ ID NO: 374 ATP-binding protein, amino acid Q5F9M1 Putative ABC transporter, INNKGTVTVGTEGTYAPFTYHDKDGK SEQ ID NO: 375 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, INNKGTVTVGTEGTYAPFTYHDKDGKLTGYDVEVTR SEQ ID NO: 376 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, VEFKETQWDSMMAGLK SEQ ID NO: 377 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, AGRFDVVANQVGLTSPER SEQ ID NO: 378 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, FDVVANQVGLTSPER SEQ ID NO: 379 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, QATFDKSEPYSWSGAVLVAHNDSNIK SEQ ID NO: 380 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, SEPYSWSGAVLVAHNDSNIK SEQ ID NO: 381 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, TAQSLTSNYGEK SEQ ID NO: 382 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, AKAAGAQLVPVDGLAQSLTLIEQK SEQ ID NO: 383 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, AAGAQLVPVDGLAQSLTLIEQK SEQ ID NO: 384 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, AAGAQLVPVDGLAQSLTLIEQKR SEQ ID NO: 385 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, RADATLNDELAVLDYLK SEQ ID NO: 386 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, RADATLNDELAVLDYLKK SEQ ID NO: 387 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, ADATLNDELAVLDYLK SEQ ID NO: 388 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, ADATLNDELAVLDYLKK SEQ ID NO: 389 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, IVWSAPADEKVGSGLIVNK SEQ ID NO: 390 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, IVWSAPADEKVGSGLIVNKGNDEAVAK SEQ ID NO: 391 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, VGSGLIVNK SEQ ID NO: 392 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, VGSGLIVNKGNDEAVAK SEQ ID NO: 393 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, FSTAINELK SEQ ID NO: 394 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, FSTAINELKADGTLK SEQ ID NO: 395 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, FSTAINELKADGTLKK SEQ ID NO: 396 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, KLGEQFFGKDISVQ SEQ ID NO: 397 periplasmic binding protein, amino acid Q5F9M1 Putative ABC transporter, LGEQFFGKDISVQ SEQ ID NO: 398 periplasmic binding protein, amino acid Q5F6Q5 Putative ABC transporter, SGYDLVVPGIAFLPR SEQ ID NO: 399 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, VNKDLIPNYKNIDPELLK SEQ ID NO: 400 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, GSNPEDLKAAAEVLK SEQ ID NO: 401 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, RFSPSIIDELAR SEQ ID NO: 402 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, FSPSIIDELAR SEQ ID NO: 403 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, ARSEEVKNNVGIEVLTPK SEQ ID NO: 404 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, SEEVKNNVGIEVLTPK SEQ ID NO: 405 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, YINYTLDPETAAK SEQ ID NO: 406 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, NGIAVTFAPASKPAR SEQ ID NO: 407 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, EKMPAELVNTR SEQ ID NO: 408 periplasmic binding protein, polyamine Q5F6Q5 Putative ABC transporter, MPAELVNTR SEQ ID NO: 409 periplasmic binding protein, polyamine Q5F7C5 Putative ABC transporter, NGNFVTYAPSSKPAR SEQ ID NO: 410 periplasmic binding protein, polyamine Q5FA28 Putative ABC transporter, SGYDLTAPSIANVGR SEQ ID NO: 411 periplasmic binding protein, polyamine Q5FA28 Putative ABC transporter, AQIPHYGNIDKDLLK SEQ ID NO: 412 periplasmic binding protein, polyamine Q5FA28 Putative ABC transporter, TGVGVWVDSFMIPR SEQ ID NO: 413 periplasmic binding protein, polyamine Q5FA28 Putative ABC transporter, YIDYTLRPEVAAK SEQ ID NO: 414 periplasmic binding protein, polyamine Q5F8K8 Putative ABC-transporter, FAEYDGYTAEAR SEQ ID NO: 415 ATP-binding protein Q5F8K8 Putative ABC-transporter, LQELQEFVAR SEQ ID NO: 416 ATP-binding protein Q5F8K8 Putative ABC-transporter, LKQADKIKSEMVEVKPSTR SEQ ID NO: 417 ATP-binding protein Q5F8K8 Putative ABC-transporter, SEMVEVKPSTR SEQ ID NO: 418 ATP-binding protein Q5F8K8 Putative ABC-transporter, LAIIGPNGAGK SEQ ID NO: 419 ATP-binding protein Q5F8B6 Putative ABC-type SREDKIQTASAPK SEQ ID NO: 420 transporter, ATP-binding protein Q5F655 Putative adenylosuccinate LLELTPALYVGK SEQ ID NO: 421 lyase Q5F655 Putative adenylosuccinate LLELTPALYVGKAEALAK SEQ ID NO: 422 lyase Q5F6V3 Putative alcohol MKMQAVVVNK SEQ ID NO: 423 dehydrogenase Q5F6V3 Putative alcohol ETGADLVVNAAKEDAAK SEQ ID NO: 424 dehydrogenase Q5F6V3 Putative alcohol TGGAHAAVVTAVSAAAFNSAVNCVR SEQ ID NO: 425 dehydrogenase Q5F6V3 Putative alcohol VVAVGLPPESMDLSIPR SEQ ID NO: 426 dehydrogenase Q5F6V3 Putative alcohol LVLDGIEVVGSLVGTR SEQ ID NO: 427 dehydrogenase Q5F6V3 Putative alcohol LVLDGIEVVGSLVGTRK SEQ ID NO: 428 dehydrogenase Q5F6V3 Putative alcohol ALDEAPAIFQEMR SEQ ID NO: 429 dehydrogenase Q5F8U5 Putative aldehyde ILAAHAGANLKK SEQ ID NO: 430 dehydrogenase Q5F758 Putative amino acid IVTVQTLGGSGALK SEQ ID NO: 431 aminotransferase Q5F6U0 Putative aminopeptidase LIEGINPSTFFK SEQ ID NO: 432 Q5F6U0 Putative aminopeptidase VVPVGTPTAEQKR SEQ ID NO: 433 Q5F6U0 Putative aminopeptidase RLEPLTEGAAK SEQ ID NO: 434 Q5FA72 Putative aminopeptidase N YLKDYQTPAYR SEQ ID NO: 435 Q5FA72 Putative aminopeptidase N LKNNVFELTIK SEQ ID NO: 436 Q5FA72 Putative aminopeptidase N RSDTLQQVQTALQHPK SEQ ID NO: 437 Q5FA72 Putative aminopeptidase N NVPHFHAQDGSGYR SEQ ID NO: 438 Q5F9D9 Putative TYHGSAMSIPVQR SEQ ID NO: 439 aminotransferases Q5F9D9 Putative AAIQVLPGR SEQ ID NO: 440 aminotransferases Q5F6R0 Putative aspartyl-tRNA LRYQVATGVR SEQ ID NO: 441 synthetase Q5F6R0 Putative aspartyl-tRNA VHPGEFFALPQSPQLFK SEQ ID NO: 442 synthetase Q5F6R0 Putative aspartyl-tRNA QVFKDALNVDLGDFPR SEQ ID NO: 443 synthetase Q5F6R0 Putative aspartyl-tRNA VVALRVPNGAK SEQ ID NO: 444 synthetase Q5F6R0 Putative aspartyl-tRNA VNDAGNLSNGEDSGLQSPIVK SEQ ID NO: 445 synthetase Q5F6R0 Putative aspartyl-tRNA YVAVHHPFTAPK SEQ ID NO: 446 synthetase Q5F6R0 Putative aspartyl-tRNA EGHEDLMVSDPANCLAR SEQ ID NO: 447 synthetase Q5F6R0 Putative aspartyl-tRNA FGFLLDNLK SEQ ID NO: 448 synthetase Q5F6R0 Putative aspartyl-tRNA FGAPPHGGLAFGLDR SEQ ID NO: 449 synthetase Q5F6R0 Putative aspartyl-tRNA LVTLMTGAESIRDVIAFPK SEQ ID NO: 450 synthetase Q5F6R0 Putative aspartyl-tRNA AQCLLTDAPNSVDDK SEQ ID NO: 451 synthetase Q5F6R0 Putative aspartyl-tRNA synthetase AQCLLTDAPNSVDDKQLR SEQ ID NO: 452 Q5FAG6 Putative atpase TLLDTVAVPNTAR SEQ ID NO: 453 Q5FAG6 Putative atpase LNVPLLGQLPLSLPVR SEQ ID NO: 454 Q5F9I6 Putative ATP-dependent QKKVIGK SEQ ID NO: 455 C1p protease Q5F8W0 Putative ATP-dependent GIDVPTITHVINYDLPK SEQ ID NO: 456 RNA helicase Q5F8W0 Putative ATP-dependent RGDHKPGKEGFGGK SEQ ID NO: 457 RNA helicase Q5FA80 Putative ATP-dependent ALAGHDLLAAAQTGTGK SEQ ID NO: 458 RNA helicase Q5FA80 Putative ATP-dependent YATASTSPAMHPVR SEQ ID NO: 459 RNA helicase Q5F697 Putative branched-chain IVLFRPTANIAR SEQ ID NO: 460 amino acid aminotranferase Q5F697 Putative branched-chain AADEIPDAPAALYLRPTLIGTDPVIGK SEQ ID NO: 461 amino acid aminotranferase Q5F697 Putative branched-chain AGSPSETALLYILASPVGDYFK SEQ ID NO: 462 amino acid aminotranferase Q5FAB9 Putative RSDNLIAR SEQ ID NO: 463 carboxypeptidase, penicillin binding protein Q5F932 Putative carboxy-terminal AVIVGTQSFGK SEQ ID NO: 464 processing protease Q5F8D2 Putative cell-division SPDVSQGQSVSDGTAVR SEQ ID NO: 465 protein Q5F8D2 Putative cell-division APHLLVAGTTGSGK SEQ ID NO: 466 protein Q5F4Z9 Putative chromosome AAQIAGLSEIPAVIK SEQ ID NO: 467 segregation protein Q5F4Z9 Putative chromosome TISDETALAMGLIENLQR SEQ ID NO: 468 segregation protein Q5F4Z9 Putative chromosome LADEFGLTHETIAQAVGK SEQ ID NO: 469 segregation protein Q5F4Z9 Putative chromosome LLSLPESVQEMLYQR SEQ ID NO: 470 segregation protein Q5F4Z9 Putative chromosome ALLTLPVVEQLELAQK SEQ ID NO: 471 segregation protein Q5F4Z9 Putative chromosome SQAALQNKRPEPK SEQ ID NO: 472 segregation protein Q5F7W9 Putative ClpB protein QRLQQHLNSLPK SEQ ID NO: 473 Q5F7W9 Putative ClpB protein VSGQGGEILPSR SEQ ID NO: 474 Q5F7W9 Putative ClpB protein EAGATEQNINAAIDAVR SEQ ID NO: 475 Q5F7W9 Putative ClpB protein TKNNPVLIGEPGVGK SEQ ID NO: 476 Q5F7W9 Putative ClpB protein LLVLDLAALIAGAK SEQ ID NO: 477 Q5F7W9 Putative ClpB protein VLVGEPSVEDTIAILR SEQ ID NO: 478 Q5F7W9 Putative ClpB protein FLPDKAIDLIDEAASR SEQ ID NO: 479 Q5F7W9 Putative ClpB protein AISDGAANIKK SEQ ID NO: 480 Q5F7W9 Putative ClpB protein IKIEQAKR SEQ ID NO: 481 Q5F7W9 Putative ClpB protein NNVGAEEIAEVVSR SEQ ID NO: 482 Q5F7W9 Putative ClpB protein SGLADPNKPYGSFLFLGPTGVGK SEQ ID NO: 483 Q5F7W9 Putative ClpB protein ALAGFLFDSEDHLIR SEQ ID NO: 484 Q5F7W9 Putative ClpB protein AIQSEIENPLAK SEQ ID NO: 485 Q5F9I4 Putative cold shock GFGFITPDEGGEDLFAHFSAINMEGFK SEQ ID NO: 486 protein Q5F9I4 Putative cold shock GFGFITPDEGGEDLFAHFSAINMEGFKTLK SEQ ID NO: 487 protein Q5F9I4 Putative cold shock VSFDVTTGPK SEQ ID NO: 488 protein Q5F9I4 Putative cold shock GKQAANIQAA SEQ ID NO: 489 protein Q5F759 Putative cytochrome IQPVGQLTMGDGIPVGER SEQ ID NO: 490 Q5F759 Putative cytochrome GGAADLTDQELKR SEQ ID NO: 491 Q5F759 Putative cytochrome AEDKGAAAPAVGVDGK SEQ ID NO: 492 Q5F759 Putative cytochrome AEDKGAAAPAVGVDGKK SEQ ID NO: 493 Q5F759 Putative cytochrome HALEGFNAMPAK SEQ ID NO: 494 Q5FAC5 Putative cytochrome SGEANPKENPELGAK SEQ ID NO: 495 Q5FAC5 Putative cytochrome MSEEDLKAVANFIQGLR SEQ ID NO: 496 Q5F716 Putative cytochrome c AATQPAPGVKPYNALQVAGR SEQ ID NO: 497 oxidase subunit Q5F716 Putative cytochrome c DVVPESNMPAFPWLAR SEQ ID NO: 498 oxidase subunit Q5F716 Putative cytochrome c NKVDVDATVANMK SEQ ID NO: 499 oxidase subunit Q5F718 Putative cytochrome c GGRGDLSDDEVK SEQ ID NO: 500 oxidase subunit Q5F598 Putative cytochrome C1 LKDIGLTDEEIKK SEQ ID NO: 501 Q5F598 Putative cytochrome C1 WFGAAPPDLTLIAR SEQ ID NO: 502 Q5F598 Putative cytochrome C1 GQPVMVKDEHGEMKPK SEQ ID NO: 503 Q5F598 Putative cytochrome C1 LYWESTGLHSR SEQ ID NO: 504 Q5F940 Putative dihydrolipoamide ELGVDLGQVK SEQ ID NO: 505 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide FGNVEVKELSR SEQ ID NO: 506 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide IKKISGQNLSR SEQ ID NO: 507 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide ISGQNLSR SEQ ID NO: 508 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide LSPLAFIIK SEQ ID NO: 509 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide AFPEFNASLDGDNLVLK SEQ ID NO: 510 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide NYFNIGFAADTPNGLVVPVIK SEQ ID NO: 511 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide NYFNIGFAADTPNGLVVPVIKDV SEQ ID NO: 512 acetyltransferase component of pyruvate dehydrogenase complex DQK Q5F940 Putative dihydrolipoamide QISQELTELSK SEQ ID NO: 513 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide QISQELTELSKK SEQ ID NO: 514 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide SQIKPVWNGKEFAPR SEQ ID NO: 515 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide VIDGAAGMR SEQ ID NO: 516 acetyltransferase component of pyruvate dehydrogenase complex Q5F940 Putative dihydrolipoamide FTVFLANLLK SEQ ID NO: 517 acetyltransferase component of pyruvate dehydrogenase complex Q5F866 Putative dihydrolipoamide LLIAAAEAR SEQ ID NO: 518 dehydrogenase Q5F866 Putative dihydrolipoamide GVPVADPLTMQTSIPHIFIAGDASNQLPLLHEAADQGK SEQ ID NO: 519 dehydrogenase Q5F942 Putative dihydrolipoamide AEAAAAPAQEAPK SEQ ID NO: 520 dehydrogenase Q5F942 Putative dihydrolipoamide TLGGVCLNVGCIPSK SEQ ID NO: 521 dehydrogenase Q5F942 Putative dihydrolipoamide ALLHNAAVIDEVR SEQ ID NO: 522 dehydrogenase Q5F942 Putative dihydrolipoamide AYKDGVVSR SEQ ID NO: 523 dehydrogenase Q5F942 Putative dihydrolipoamide LTGGLAGMAK SEQ ID NO: 524 dehydrogenase Q5F942 Putative dihydrolipoamide NCIIAAGSR SEQ ID NO: 525 dehydrogenase Q5F942 Putative dihydrolipoamide IIDSSGALALKEVPGK SEQ ID NO: 526 dehydrogenase Q5F942 Putative dihydrolipoamide EPQRYDAVLVAAGR SEQ ID NO: 527 dehydrogenase Q5F942 Putative dihydrolipoamide APNGKLISAEK SEQ ID NO: 528 dehydrogenase Q5F942 Putative dihydrolipoamide TNVPHIYAIGDIVGQPMLAHK SEQ ID NO: 529 dehydrogenase Q5F942 Putative dihydrolipoamide AVHEGHVAAENCAGHK SEQ ID NO: 530 dehydrogenase Q5F942 Putative dihydrolipoamide VIPGVAYTSPEVAWVGETELSAK SEQ ID NO: 531 dehydrogenase Q5F942 Putative dihydrolipoamide dehydrogenase AIANGCDNGFTK SEQ ID NO: 532 Q5F875 Putative dihydrolipoamide succinyltransferase E2 IDTAATVAAEAPAAAPAEAAPAAVPAAAQNNAAMPAAAK SEQ ID NO: 533 component Q5F875 Putative dihydrolipoamide LAAETGVDVNVLQGSGR SEQ ID NO: 534 succinyltransferase E2 component Q5F875 Putative dihydrolipoamide VLKEDVQNAAAKPAAAVAPAVALPAGARPEER SEQ ID NO: 535 succinyltransferase E2 component Q5F875 Putative dihydrolipoamide VLKEDVQNAAAKPAAAVAPAVALPAGARPEERVPMSR SEQ ID NO: 536 succinyltransferase E2 component Q5F875 Putative dihydrolipoamide LLASQQENAILTTFNEVNMKPIMDLR SEQ ID NO: 537 succinyltransferase E2 component Q5F875 Putative dihydrolipoamide EAVLTLVAIKDALEDPVR SEQ ID NO: 538 succinyltransferase E2 component Q5F898 Putative D-lactate AALGEFESLPVSGEYIHR SEQ ID NO: 539 dehydrogenase Q5F898 Putative D-lactate FGTHQLPK SEQ ID NO: 540 dehydrogenase Q5F898 Putative D-lactate FLPDHLPK SEQ ID NO: 541 dehydrogenase Q5F898 Putative D-lactate FAVASAAIR SEQ ID NO: 542 dehydrogenase Q5F898 Putative D-lactate GAQYPAEHNVGHLYEAKPALK SEQ ID NO: 543 dehydrogenase Q5F898 Putative D-lactate KLDPTNSFNPGIGK SEQ ID NO: 544 dehydrogenase Q5F533 Putative DNA polymerase I GGISTNEAVLEQLAPDYPLPK SEQ ID NO: 545 Q5F533 Putative DNA polymerase I LASNNPNLQNIPIR SEQ ID NO: 546 Q5F533 Putative DNA polymerase I AAINAPMQGTASDLIKR SEQ ID NO: 547 Q5F8J6 Putative DNA-binding MNKSELIEAIAQEADISK SEQ ID NO: 548 protein Hu Q5F8J6 Putative DNA-binding MNKSELIEAIAQEADISKAAAQK SEQ ID NO: 549 protein Hu Q5F8J6 Putative DNA-binding ALDATTNAVTNALK SEQ ID NO: 550 protein Hu Q5F8J6 Putative DNA-binding TGEPLTIAAAK SEQ ID NO: 551 protein Hu Q5F5I8 Putative electron transfer QLSHSDRPELTQAK SEQ ID NO: 552 flavoprotein alpha-subunit Q5F5I9 Putative electron transfer AVADKENPQIFFLGK SEQ ID NO: 553 flavoprotein beta-subunit Q5F5I9 Putative electron transfer KKPLEKLDSADLATDISPR SEQ ID NO: 554 flavoprotein beta-subunit Q5F5I9 Putative electron transfer LDSADLATDISPR SEQ ID NO: 555 flavoprotein beta-subunit Q5F5I9 Putative electron transfer VASVAELVEK SEQ ID NO: 556 flavoprotein beta-subunit Q5F8S3 Putative ferredoxin- IILVHGVR SEQ ID NO: 557 NADP reductase Q5F8S3 Putative ferredoxin- TGVRGDYLIER SEQ ID NO: 558 NADP reductase Q5FAI4 Putative fructose- QLLDHAAENSYGLPAFNVNNLEQMR SEQ ID NO: 559 bisphosphate aldolase Q5FAI4 Putative fructose- AIMEAADQVNAPVIVQASAGAR SEQ ID NO: 560 bisphosphate aldolase Q5FAI4 Putative fructose- KYAGAPFLR SEQ ID NO: 561 bisphosphate aldolase Q5FAI4 Putative fructose- LSHDQMLTSVEDAVR SEQ ID NO: 562 bisphosphate aldolase Q5FAI4 Putative fructose- FTRPPTGDVLR SEQ ID NO: 563 bisphosphate aldolase Q5FAI4 Putative fructose- VINEYGGNIGETYGVPVEEIVEGIK SEQ ID NO: 564 bisphosphate aldolase Q5FAI4 Putative fructose- KVNIDTDLR SEQ ID NO: 565 bisphosphate aldolase Q5FAI4 Putative fructose- LASTGAVRR SEQ ID NO: 566 bisphosphate aldolase Q5F5J3 Putative glyceraldehyde 3- FQGTAELKDDAIVVNGR SEQ ID NO: 567 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- KVVISAPGGNDVK SEQ ID NO: 568 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- AAALNIVPNSTGAAK SEQ ID NO: 569 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- AIGLVIPELNGK SEQ ID NO: 570 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- AIGLVIPELNGKLDGSAQR SEQ ID NO: 571 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- VPVATGSLTELVSVLERPATK SEQ ID NO: 572 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- VPVATGSLTELVSVLERPATKEEINAAMK SEQ ID NO: 573 phosphate dehydrogenase C Q5F5J3 Putative glyceraldehyde 3- TLEYFAGKI SEQ ID NO: 574 phosphate dehydrogenase C Q5F8H0 Putative GTP-binding NIAIIAHVDHGK SEQ ID NO: 575 protein Q5F8H0 Putative GTP-binding VMDSNDLEKER SEQ ID NO: 576 protein Q5F8H0 Putative GTP-binding ALALGLKPIVVINK SEQ ID NO: 577 protein Q5F8H0 Putative GTP-binding LEETDESSDMRPLFDTILK SEQ ID NO: 578 protein Q5F8H0 Putative GTP-binding IKPGQTVAVMNHEQQIAQGR SEQ ID NO: 579 protein Q5F8H0 Putative GTP-binding INQLLGFK SEQ ID NO: 580 protein Q5F8H0 Putative GTP-binding EGYELAVGKPR SEQ ID NO: 581 protein Q5F8H0 Putative GTP-binding TRLEYHIPAR SEQ ID NO: 582 protein Q5F8H0 Putative GTP-binding LEYHIPAR SEQ ID NO: 583 protein Q5FAG0 Putative GTP-binding MQPAIVEFVDIAGLVAGASK SEQ ID NO: 584 protein Q5FAG0 Putative GTP-binding KLLPHLDEGKPVR SEQ ID NO: 585 protein Q5F7T8 Putative isocitrate STIVYTHTDEAPALATQSLLPIVQAFAR SEQ ID NO: 586 dehydrogenase Q5F7T8 Putative isocitrate ILAAFPEYLTEAQR SEQ ID NO: 587 dehydrogenase Q5F7T8 Putative isocitrate LPNISASVPQLTAAIK SEQ ID NO: 588 dehydrogenase Q5F7T8 Putative isocitrate IKGSAVNPVLR SEQ ID NO: 589 dehydrogenase Q5F7T8 Putative isocitrate GSAVNPVLR SEQ ID NO: 590 dehydrogenase Q5F7T8 Putative isocitrate DAPVKDWVQLAVNR SEQ ID NO: 591 dehydrogenase Q5F7T8 Putative isocitrate LSNTPAVFWLDENRPHDK SEQ ID NO: 592 dehydrogenase Q5F7T8 Putative isocitrate LKNGEDTISVTGNVLR SEQ ID NO: 593 dehydrogenase Q5F7T8 Putative isocitrate DYLTDLFPILELGTSAK SEQ ID NO: 594 dehydrogenase Q5F7T8 Putative isocitrate HVQQFLEENHLR SEQ ID NO: 595 dehydrogenase Q5F7T8 Putative isocitrate AQVLADTLDAATEK SEQ ID NO: 596 dehydrogenase Q5F7T8 Putative isocitrate AAFAPLAAALTADEAK SEQ ID NO: 597 dehydrogenase Q5F7T8 Putative isocitrate AAFAPLAAALTADEAKIVEELSAVQGK SEQ ID NO: 598 dehydrogenase Q5F7T8 Putative isocitrate AAQAMRPSATFNQVLNAL SEQ ID NO: 599 dehydrogenase Q5F8Q4 Putative khg/kdpg 4- TPVGLEAIR SEQ ID NO: 600 hydroxy-2-oxoglutarate aldolase Q5F8Q4 Putative khg/kdpg 4- AVEDAGAVFAISPGLHESLAR SEQ ID NO: 601 hydroxy-2-oxoglutarate aldolase Q5F8X1 Putative L-lactate NVGDLSSLSSWTAEQFDPR SEQ ID NO: 602 dehydrogenase Q5F8X1 Putative L-lactate SGADALVVSNHGGR SEQ ID NO: 603 dehydrogenase Q5F9Z4 Putative malic enzyme, FHELPVPGK SEQ ID NO: 604 malate dehydrogenase/ oxaloacetate- decarboxylating Q5F9Z4 Putative malic enzyme, ETRPDVVIGTGR SEQ ID NO: 605 malatedehydrogenase/ oxaloacetate- decarboxylating Q5F9Z4 Putative malic enzyme, GALDVGATTINEEMKR SEQ ID NO: 606 malatedehydrogenase/ oxaloacetate- decarboxylating Q5F9Z4 Putative malic enzyme, AAMESGVATRPIADLEAYAAK SEQ ID NO: 607 malatedehydrogenase/ oxaloacetate- decarboxylating Q5F798 Putative N utilisation EQNLNEFLAVKEDIVSGTVK SEQ ID NO: 608 substance protein A Q5F798 Putative N utilisation HGIIVEVVAGK SEQ ID NO: 609 substance protein A Q5F798 Putative N utilisation IDPQGTCIGVR SEQ ID NO: 610 substance protein A Q5F798 Putative N utilisation VNAVSNELSGER SEQ ID NO: 611 substance protein A Q5F798 Putative N utilisation LGEVSDDMR SEQ ID NO: 612 substance protein A Q5F798 Putative N utilisation NLEGVDADMLLSLAEAGITTR SEQ ID NO: 613 substance protein A Q5F621 Putative NADH GSAPLQETSHAAVPAAR SEQ ID NO: 614 dehydrogenase I chain Q5F621 Putative NADH GLGLQDGQTAVAK SEQ ID NO: 615 dehydrogenase I chain Q5F7J5 Putative nitrogen IGDGKIFVLPVEEAIR SEQ ID NO: 616 regulatory protein P-II Q5F7J5 Putative nitrogen IFVLPVEEAIR SEQ ID NO: 617 regulatory protein P-II Q5F8K6 Putative peptidyl-prolyl LPAHEAKPSFEQEK SEQ ID NO: 618 cis-trans isomerase Q5F8K6 Putative peptidyl-prolyl AKPANGKPAYVR SEQ ID NO: 619 cis-trans isomerase Q5F8K6 Putative peptidyl-prolyl QALAQQQSANTFDLLIR SEQ ID NO: 620 cis-trans isomerase Q5F865 Putative peroxiredoxin ALQDRTGQKVPSVVFR SEQ ID NO: 621 family protein/glutaredoxin Q5F865 Putative peroxiredoxin TGQKVPSVVFR SEQ ID NO: 622 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VPSVVFR SEQ ID NO: 623 family protein/glutaredoxin Q5F865 Putative peroxiredoxin TRVGDTWKDVSTDDLFK SEQ ID NO: 624 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VGDTWKDVSTDDLFK SEQ ID NO: 625 family protein/glutaredoxin Q5F865 Putative peroxiredoxin KVVVFSLPGAFTPTCSSSHLPR SEQ ID NO: 626 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VVVFSLPGAFTPTCSSSHLPR SEQ ID NO: 627 family protein/glutaredoxin Q5F865 Putative peroxiredoxin EDLGFGKR SEQ ID NO: 628 family protein/glutaredoxin Q5F865 Putative peroxiredoxin YSMLVNDGVVEK SEQ ID NO: 629 family protein/glutaredoxin Q5F865 Putative peroxiredoxin MFIEPEEPGDPFK SEQ ID NO: 630 family protein/glutaredoxin Q5F865 Putative peroxiredoxin MFIEPEEPGDPFKVSDADTMLK SEQ ID NO: 631 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VSDADTMLK SEQ ID NO: 632 family protein/glutaredoxin Q5F865 Putative peroxiredoxin FVAPDWK SEQ ID NO: 633 family protein/glutaredoxin Q5F865 Putative peroxiredoxin FVAPDWKAQESVAIFTKPGCQFCAK SEQ ID NO: 634 family protein/glutaredoxin Q5F865 Putative peroxiredoxin AQESVAIFTKPGCQFCAK SEQ ID NO: 635 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VKQALQDK SEQ ID NO: 636 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VKQALQDKGLSYEEIVLGK SEQ ID NO: 637 family protein/glutaredoxin Q5F865 Putative peroxiredoxin VKQALQDKGLSYEEIVLGKDATVTSVR SEQ ID NO: 638 family protein/glutaredoxin Q5F865 Putative peroxiredoxin QALQDKGLSYEEIVLGK SEQ ID NO: 639 family protein/glutaredoxin Q5F865 Putative peroxiredoxin QALQDKGLSYEEIVLGKDATVTSVR SEQ ID NO: 640 family protein/glutaredoxin Q5F865 Putative peroxiredoxin GLSYEEIVLGKDATVTSVR SEQ ID NO: 641 family protein/glutaredoxin Q5F865 Putative peroxiredoxin AITGKMTAPQVFIGGK SEQ ID NO: 642 family protein/glutaredoxin Q5F865 Putative peroxiredoxin MTAPQVFIGGK SEQ ID NO: 643 family protein/glutaredoxin Q5F865 Putative peroxiredoxin YIGGSEDLEAYLAK SEQ ID NO: 644 family protein/glutaredoxin Q5F865 Putative peroxiredoxin YIGGSEDLEAYLAKN SEQ ID NO: 645 family protein/glutaredoxin Q5F8Q3 Putative NQLGCSNLAHGYAAMPK SEQ ID NO: 646 phosphogluconate dehydratase Q5F8Q3 Putative YAAGHLAR SEQ ID NO: 647 phosphogluconate dehydratase Q5F8Q3 Putative ADVNHFTAAGGLPFVIR SEQ ID NO: 648 phosphogluconate dehydratase Q5F8Q3 Putative KADNPFSPDGGLR SEQ ID NO: 649 phosphogluconate dehydratase Q5F8Q3 Putative LTPPLGILQDR SEQ ID NO: 650 phosphogluconate dehydratase Q5F8Q3 Putative VPASIHMTPEALMGGNIAK SEQ ID NO: 651 phosphogluconate dehydratase Q5FA20 Putative VLVVPVSAGLNTSAAAQAFAK SEQ ID NO: 652 phosphotransacetylase Q5FA20 Putative VAALDAANLVIEGIAPDADKIYLAGK SEQ ID NO: 653 phosphotransacetylase Q5FA20 Putative TGLTFFGSSDALKDVSVLAGR SEQ ID NO: 654 phosphotransacetylase Q5FA20 Putative DVSVLAGR SEQ ID NO: 655 phosphotransacetylase Q5FA20 Putative RLSPAQFR SEQ ID NO: 656 phosphotransacetylase Q5FA20 Putative IVLPEGAEPR SEQ ID NO: 657 phosphotransacetylase Q5FA20 Putative TVQAAAICHEK SEQ ID NO: 658 phosphotransacetylase Q5FA20 Putative TVQAAAICHEKGIAR SEQ ID NO: 659 phosphotransacetylase Q5FA20 Putative REEVEAVAKER SEQ ID NO: 660 phosphotransacetylase Q5FA20 Putative EKRPDLAIDGPLQYDAATVPGVGK SEQ ID NO: 661 phosphotransacetylase Q5FA20 Putative SKAPGSPVAGQATVLVFPDLNTGNCTYK SEQ ID NO: 662 phosphotransacetylase Q5FA20 Putative SANVLSVGPLLQGLR SEQ ID NO: 663 phosphotransacetylase Q5FAC8 Putative pilus assembly KTDAKLPK SEQ ID NO: 664 protein Q5FAC8 Putative pilus assembly YVIAKLPK SEQ ID NO: 665 protein Q5FAC8 Putative pilus assembly NIVASVPQNLATIEQLTYTAK SEQ ID NO: 666 protein Q5FAC8 Putative pilus assembly GIAQTVASQTNADVQCVHPAR SEQ ID NO: 667 protein Q5FAC8 Putative pilus assembly YFANDLKTDEQQFELDAPTLTK SEQ ID NO: 668 protein Q5FAC9 Putative pilus assembly ILDSLNEAVPGSTYLTSLDAVTADSYR SEQ ID NO: 669 protein Q5FAC9 Putative pilus assembly AAESKENPASGNAQEAN SEQ ID NO: 670 protein Q5F5Y3 Putative ribonuclease E FQIEHQIESAFSR SEQ ID NO: 671 Q5F5Y3 Putative ribonuclease E LKPALGESSHAACPR SEQ ID NO: 672 Q5F5Y3 Putative ribonuclease E GIESTALHVLR SEQ ID NO: 673 Q5F5Y3 Putative ribonuclease E AQVPVDVATFLLNEK SEQ ID NO: 674 Q5F5Y3 Putative ribonuclease E AQVPVDVATFLLNEKR SEQ ID NO: 675 Q5F5Y3 Putative ribonuclease E IRTDDVEEDGEPSYKR SEQ ID NO: 676 Q5F5Y3 Putative ribonuclease E VAEPEEDESAKPFGGEK SEQ ID NO: 677 Q5F5Y3 Putative ribonuclease E AARPEPAVK SEQ ID NO: 678 Q5F5Y3 Putative ribonuclease E HTSPAPTAAPEK SEQ ID NO: 679 Q5F5Y3 Putative ribonuclease E IFGGSETQAVPAAETSEKR SEQ ID NO: 680 Q5F5Y3 Putative ribonuclease E NVQPAAPVADAAPPETEGQTGKR SEQ ID NO: 681 Q5F5Y3 Putative ribonuclease E IEQYLNIHDTADKVR SEQ ID NO: 682 Q5F5Y3 Putative ribonuclease E AWAAQPEVQAGR SEQ ID NO: 683 Q5F870 Putative succinate IYQRPFGGHTAEHGK SEQ ID NO: 684 dehydrogenase flavoprotein subunit Q5F870 Putative succinate IDHIGAEK SEQ ID NO: 685 dehydrogenase flavoprotein subunit Q5F870 Putative succinate SVQLHAGVFR SEQ ID NO: 686 dehydrogenase flavoprotein subunit Q5F649 Putative thiol: disulphide TEHVVWQK SEQ ID NO: 687 interchange protein Q5F649 Putative thiol: disulphide LAAAVDMAAAESK SEQ ID NO: 688 interchange protein Q5F649 Putative thiol: disulphide LAAAVDMAAAESKDVANSHIFDAMVNQK SEQ ID NO: 689 interchange protein Q5F649 Putative thiol: disulphide DVANSHIFDAMVNQK SEQ ID NO: 690 interchange protein Q5F649 Putative thiol: disulphide IKLQEPEVLKK SEQ ID NO: 691 interchange protein Q5F649 Putative thiol: disulphide VLAAYESPESQAR SEQ ID NO: 692 interchange protein Q5F8S1 Putative threonine AQMYSLQDGNIHNIAVK SEQ ID NO: 693 synthase Q5F8S1 Putative threonine NSAHTYVTSSPSMDISK SEQ ID NO: 694 synthase Q5F7Y3 Putative transketolase FLNHNPANPK SEQ ID NO: 695 Q5F7Y3 Putative transketolase AETGKPSIICCK SEQ ID NO: 696 Q5F7Y3 Putative transketolase THGAPLGADEIEATRK SEQ ID NO: 697 Q5F7Y3 Putative transketolase LEAGWNELFAQYQAK SEQ ID NO: 698 Q5F7Y3 Putative transketolase YPAEAAEFVR SEQ ID NO: 699 Q5F7Y3 Putative transketolase KLPENFDEYVQTALK SEQ ID NO: 700 Q5F7Y3 Putative transketolase KASQNSIEILAK SEQ ID NO: 701 Q5F7Y3 Putative transketolase DKGGNYIHYGVR SEQ ID NO: 702 Q5F7Y3 Putative transketolase VLAGQGIAVR SEQ ID NO: 703 Q5F7Y3 Putative transketolase YVGLNGAVVGINR SEQ ID NO: 704 Q5F7Y3 Putative transketolase AFGFTVDNVVDTVK SEQ ID NO: 705 Q5F5L4 Putative twitching INSALTPQPQK SEQ ID NO: 706 motility-like protein Q5F5L4 Putative twitching ELGLPEKLKDLAVAPR SEQ ID NO: 707 motility-like protein Q5F9P7 Putative twitching GSDLFVTTHFPPAMK SEQ ID NO: 708 motility-like protein Q5F9P7 Putative twitching LDGKITR SEQ ID NO: 709 motility-like protein Q5F9P7 Putative twitching ITDEPLTAEK SEQ ID NO: 710 motility-like protein Q5F5Q1 Putative two-component IGAIDFLEKPISLQK SEQ ID NO: 711 system transcriptional response regulator Q5FA55 Putative two-component STVPIIMLTAK SEQ ID NO: 712 system transcriptional response regulator Q5FA55 Putative two-component INAILRR SEQ ID NO: 713 system transcriptional response regulator Q5FA55 Putative two-component AQHSGEQNNAPNSISVSDVVLYPAKR SEQ ID NO: 714 system transcriptional response regulator Q5FA55 Putative two-component LGDASLIQTVR SEQ ID NO: 715 system transcriptional response regulator Q5F4X2 Putative uncharacterized VQWLDPVPEVLR SEQ ID NO: 716 protein Q5F537 Putative uncharacterized FGVIQTGLQLQGKPQSAPPTQK SEQ ID NO: 717 protein Q5F573 Putative uncharacterized AAAEHPLSVQNEER SEQ ID NO: 718 protein Q5F573 Putative uncharacterized KVNINIPFPQR SEQ ID NO: 719 protein Q5F5A6 Putative uncharacterized IVIMAALNVVHDLLK SEQ ID NO: 720 protein Q5F5E4 Putative uncharacterized SEQAAEGVYNYITVASLPR SEQ ID NO: 721 protein Q5F5P4 Putative uncharacterized QGDTLWGISGK SEQ ID NO: 722 protein Q5F5P4 Putative uncharacterized YLYSPWQWCR SEQ ID NO: 723 protein Q5F5P4 Putative uncharacterized LWGANRDQIHNPDLIYPGQVLVLR SEQ ID NO: 724 protein Q5F5P4 Putative uncharacterized DQIHNPDLIYPGQVLVLR SEQ ID NO: 725 protein Q5F5P4 Putative uncharacterized ISPDKEVSGYGIPAIDVNFYR SEQ ID NO: 726 protein Q5F5P4 Putative uncharacterized VFMQHPQIVSR SEQ ID NO: 727 protein Q5F5P4 Putative uncharacterized VFMQHPQIVSRK SEQ ID NO: 728 protein Q5F5P4 Putative uncharacterized VFMQHPQIVSRKETAAAPR SEQ ID NO: 729 protein Q5F5P4 Putative uncharacterized LLSGPEGR SEQ ID NO: 730 protein Q5F5P4 Putative uncharacterized INKNITDPDTGKFLGQEVAFSGIVR SEQ ID NO: 731 protein Q5F5P4 Putative uncharacterized NITDPDTGKFLGQEVAFSGIVR SEQ ID NO: 732 protein Q5F5P4 Putative uncharacterized FLGQEVAFSGIVR SEQ ID NO: 733 protein Q5F5P4 Putative uncharacterized SLDYTDSALEQR SEQ ID NO: 734 protein Q5F5P4 Putative uncharacterized LKDNEYYTR SEQ ID NO: 735 protein Q5F5P4 Putative uncharacterized SIQPLVVETAISEIQQGDYLMK SEQ ID NO: 736 protein Q5F5P4 Putative uncharacterized IVSVFEGVGVGGQFK SEQ ID NO: 737 protein Q5F5P4 Putative uncharacterized TITIDKGGDDGLDKGAVLSLYKR SEQ ID NO: 738 protein Q5F5P4 Putative uncharacterized KKTMQVNLSNNLTEEPK SEQ ID NO: 739 protein Q5F5P4 Putative uncharacterized TMQVNLSNNLTEEPK SEQ ID NO: 740 protein Q5F5P4 Putative uncharacterized SRDTVELISTPAEEVGLAMVYHTAPK SEQ ID NO: 741 protein Q5F5P4 Putative uncharacterized DTVELISTPAEEVGLAMVYHTAPK SEQ ID NO: 742 protein Q5F5P4 Putative uncharacterized DLDNMPDQGR SEQ ID NO: 743 protein Q5F5W7 Putative uncharacterized IYLESKQARNIQK SEQ ID NO: 744 protein Q5F653 Putative uncharacterized TILHAPDKQSLTYK SEQ ID NO: 745 protein Q5F6A2 Putative uncharacterized LLTAGPNLLPDNPER SEQ ID NO: 746 protein Q5F6A4 Putative uncharacterized IDSSVIDAQVAAFR SEQ ID NO: 747 protein Q5F6A4 Putative uncharacterized AEDTPQLR SEQ ID NO: 748 protein Q5F6A4 Putative uncharacterized QSLLENEVVNTVVAQEVKR SEQ ID NO: 749 protein Q5F6A4 Putative uncharacterized SAEFKDALAK SEQ ID NO: 750 protein Q5F6A4 Putative uncharacterized KSGDDKKPSFK SEQ ID NO: 751 protein Q5F6K2 Putative uncharacterized VAPKPTPEQILNSGSIEK SEQ ID NO: 752 protein Q5F6M9 Putative uncharacterized AATPGPYTFILQATK SEQ ID NO: 753 protein Q5F6R9 Putative uncharacterized SAKNNGNVQR SEQ ID NO: 754 protein Q5F6V1 Putative uncharacterized RIMVGKNK SEQ ID NO: 755 protein Q5F755 Putative uncharacterized FKPLALGIDQDLIAALPQYDSALIAR SEQ ID NO: 756 protein Q5F7C9 Putative uncharacterized AGATDSEIAGALATAIALNAGAAYTYALR SEQ ID NO: 757 protein Q5F7C9 Putative uncharacterized ALEAVETQK SEQ ID NO: 758 protein Q5F7D9 Putative uncharacterized TEQETLQTIPSPK SEQ ID NO: 759 protein Q5F7E1 Putative uncharacterized VVFVSIDPERDTPEIIGK SEQ ID NO: 760 protein Q5F7E1 Putative uncharacterized QFNPDFIGLTATGGQNLPVIK SEQ ID NO: 761 protein Q5F7E4 Putative uncharacterized IVALVTVKPEYTETLAAQFKELVK SEQ ID NO: 762 protein Q5F7F5 Putative uncharacterized SVQINGQAAKP SEQ ID NO: 763 protein Q5F7W0 Putative uncharacterized VKPAGYAAPK SEQ ID NO: 764 protein Q5F7W0 Putative uncharacterized TAAVESRPAVPAAAQTPVKPAAQPPVQSAPQPAAPAAENK SEQ ID NO: 765 protein Q5F7W0 Putative uncharacterized AVPAPAPAPQSPAASPSGTR SEQ ID NO: 766 protein Q5F7W0 Putative uncharacterized SVGGIVWQRPTQGK SEQ ID NO: 767 protein Q5F7X2 Putative uncharacterized ASAEEAVTEAK SEQ ID NO: 768 protein Q5F7X2 Putative uncharacterized ASAEEAVTEAKDAAAETK SEQ ID NO: 769 protein Q5F7X2 Putative uncharacterized ASAEEAVTEAKDAAAETKEAVSEAAKDTLNK SEQ ID NO: 770 protein Q5F7X2 Putative uncharacterized EAVSEAAKDTLNK SEQ ID NO: 771 protein Q5F7X2 Putative uncharacterized EAVSEAAKDTLNKAADAAQEAADKMKDAAK SEQ ID NO: 772 protein Q5F7X2 Putative uncharacterized AADAAQEAADKMK SEQ ID NO: 773 protein Q5F7X2 Putative uncharacterized AADAAQEAADKMKDAAK SEQ ID NO: 774 protein Q5F839 Putative uncharacterized HLVVAVDGSETSINALK SEQ ID NO: 775 protein Q5F839 Putative uncharacterized HAAELAGVNGAR SEQ ID NO: 776 protein Q5F839 Putative uncharacterized LTLVHVANPAEYMALAPEFLQHESYEAAAVAQGNEVLDAAER SEQ ID NO: 777 protein Q5F848 Putative uncharacterized TPAVQQPADAEVLK SEQ ID NO: 778 protein Q5F848 Putative uncharacterized KPTLPAANEMAR SEQ ID NO: 779 protein Q5F848 Putative uncharacterized APNESNAVTEQKPGLFKR SEQ ID NO: 780 protein Q5F883 Putative uncharacterized LRVEAQR SEQ ID NO: 781 protein Q5F884 Putative uncharacterized NILLPLATEHGQIAR SEQ ID NO: 782 protein Q5F884 Putative uncharacterized AALAGSNIDPIAFER SEQ ID NO: 783 protein Q5F885 Putative uncharacterized IAKDEPDMPRPK SEQ ID NO: 784 protein Q5F8C2 Putative uncharacterized LFDEHNELDDKITGLANNPVTSGAETIDELKK SEQ ID NO: 785 protein Q5F8C2 Putative uncharacterized LKLKDELYAILQK SEQ ID NO: 786 protein Q5F8C4 Putative uncharacterized ANENSPNIYFIR SEQ ID NO: 787 protein Q5F8D7 Putative uncharacterized IDDLGSTVQGR SEQ ID NO: 788 protein Q5F8D7 Putative uncharacterized LLDSQDPTAR SEQ ID NO: 789 protein Q5F8D7 Putative uncharacterized ATFYIVPNMNPDGSALGNLR SEQ ID NO: 790 protein Q5F8Q1 Putative uncharacterized SPIAFFNALSQK SEQ ID NO: 791 protein Q5F8Q1 Putative uncharacterized IVPTNHADSNTGLVR SEQ ID NO: 792 protein Q5F8T2 Putative uncharacterized NLDKTQAAAER SEQ ID NO: 793 protein Q5F8T2 Putative uncharacterized AAEQTGNAVEKGWDK SEQ ID NO: 794 protein Q5F8T2 Putative uncharacterized AAEQTGNAVEKGWDKTK SEQ ID NO: 795 protein Q5F8T2 Putative uncharacterized AAEQTGNAVEKGWDKTKEAVK SEQ ID NO: 796 protein Q5F8T2 Putative uncharacterized AAEQTGNAVEKGWDKTKEAVKK SEQ ID NO: 797 protein Q5F933 Putative uncharacterized AAVAAATNDVENKK SEQ ID NO: 798 protein Q5F933 Putative uncharacterized KQGVTDAAEQTESR SEQ ID NO: 799 protein Q5F933 Putative uncharacterized QGVTDAAEQTESR SEQ ID NO: 800 protein Q5F987 Putative uncharacterized LKQPRRR SEQ ID NO: 801 protein Q5F9G1 Putative uncharacterized GVAVINHPNPLQGGTNTNK SEQ ID NO: 802 protein Q5F9H4 Putative uncharacterized LNFGQIGSHIAGDGAVR SEQ ID NO: 803 protein Q5F9H$ Putative uncharacterized LGSLQEQRAPR SEQ ID NO: 804 protein Q5FAB8 Putative uncharacterized NAVDIGSKPNADVAWK SEQ ID NO: 805 protein Q5FAB8 Putative uncharacterized NKPAGIISHSVGK SEQ ID NO: 806 protein Q5FA46 Putative zinc-binding VGQHVVVEPYIIRDDVPTGEGSNYHLSK SEQ ID NO: 807 alcohol dehydrogenas Q5FA46 Putative zinc-binding DMNFIGLGGCGGGLSEK SEQ ID NO: 808 alcohol dehydrogenas Q5FA46 Putative zinc-binding AGDVALVGGAGPIGLLLAAVLK SEQ ID NO: 809 alcohol dehydrogenas Q5FA46 Putative zinc-binding IKLDKLVSEGFER SEQ ID NO: 810 alcohol dehydrogenas Q5FA46 Putative zinc-binding LIHNNESAVK SEQ ID NO: 811 alcohol dehydrogenas Q5F939 Pyruvate dehydrogenase AQYLLENLLK SEQ ID NO: 812 E1 component Q5F939 Pyruvate dehydrogenase MPHGTTTPYLNTVSVENEK SEQ ID NO: 813 E1 component Q5F939 Pyruvate dehydrogenase LTEDQLNNFR SEQ ID NO: 814 E1 component Q5F939 Pyruvate dehydrogenase EGLDNLIFVINCNLQR SEQ ID NO: 815 E1 component Q5F939 Pyruvate dehydrogenase IIQELEGNFAGAGWNVVK SEQ ID NO: 816 E1 component Q5F939 Pyruvate dehydrogenase LLAKDKDGILR SEQ ID NO: 817 E1 component Q5F939 Pyruvate dehydrogenase DGAYVREHFFNTPELK SEQ ID NO: 818 E1 component Q5F939 Pyruvate dehydrogenase ALVADMTDEQLWALNR SEQ ID NO: 819 E1 component Q5F939 Pyruvate dehydrogenase GGHDPQKVYNAYDR SEQ ID NO: 820 E1 component Q5F939 Pyruvate dehydrogenase AANHADGKPTVILAK SEQ ID NO: 821 E1 component Q5F939 Pyruvate dehydrogenase TIKGYGMGASGEGQNVAHQAK SEQ ID NO: 822 E1 component Q5F939 Pyruvate dehydrogenase GYGMGASGEGQNVAHQAK SEQ ID NO: 823 E1 component Q5F939 Pyruvate dehydrogenase ASLKQFR SEQ ID NO: 824 E1 component Q5F939 Pyruvate dehydrogenase RDALGGYLPQR SEQ ID NO: 825 E1 component Q5F939 Pyruvate dehydrogenase DALGGYLPQR SEQ ID NO: 826 E1 component Q5F939 Pyruvate dehydrogenase KPTQEVLEVPELSAFDAQLK SEQ ID NO: 827 E1 component Q5F939 Pyruvate dehydrogenase SSGEREFSTTMAFVR SEQ ID NO: 828 E1 component Q5F939 Pyruvate dehydrogenase ILSTLLKDKK SEQ ID NO: 829 E1 component Q5F939 Pyruvate dehydrogenase TFGMEGMFR SEQ ID NO: 830 E1 component Q5F939 Pyruvate dehydrogenase GQQYTPQDKDQLMFYK SEQ ID NO: 831 E1 component Q5F939 Pyruvate dehydrogenase IGDLAWAAGDMHAR SEQ ID NO: 832 E1 component Q5F939 Pyruvate dehydrogenase KVQLMGSGTILQEVIAGAELLK SEQ ID NO: 833 E1 component Q5F939 Pyruvate dehydrogenase VQLMGSGTILQEVIAGAELLK SEQ ID NO: 834 E1 component Q5F939 Pyruvate dehydrogenase ADFGVEADIWSCPSFNLLHR SEQ ID NO: 835 E1 component Q5F939 Pyruvate dehydrogenase LHPLEAEKVPFVTSQLQGHDGPVIAATDYIR SEQ ID NO: 836 E1 component Q5F939 Pyruvate dehydrogenase VPFVTSQLQGHDGPVIAATDYIR SEQ ID NO: 837 E1 component Q5F939 Pyruvate dehydrogenase AYIPNDYHVLGTDGFGR SEQ ID NO: 838 E1 component Q5F939 Pyruvate dehydrogenase FFEVDRYNVAVAALAALAEQGK SEQ ID NO: 839 E1 component Q5F939 Pyruvate dehydrogenase YNVAVAALAALAEQGK SEQ ID NO: 840 E1 component Q5F939 Pyruvate dehydrogenase YNVAVAALAALAEQGKVSK SEQ ID NO: 841 E1 component Q5F939 Pyruvate dehydrogenase VSKETVQQAIEK SEQ ID NO: 842 E1 component Q5F939 Pyruvate dehydrogenase YGIKADSAPSWK SEQ ID NO: 843 E1 component Q5F939 Pyruvate dehydrogenase YGIKADSAPSWKR SEQ ID NO: 844 E1 component Q5F939 Pyruvate dehydrogenase ADSAPSWKR SEQ ID NO: 845 E1 component Q5F5N5 Pyruvate kinase IVATLGPGSNNVELLEDMIR SEQ ID NO: 846 Q5F5N5 Pyruvate kinase VGGLNVVR SEQ ID NO: 847 Q5F5N5 Pyruvate kinase FNFSHGTPEFHQENAR SEQ ID NO: 848 Q5F5N5 Pyruvate kinase GGGLSAGALTEKDFR SEQ ID NO: 849 Q5F5N5 Pyruvate kinase GSTAVRPGLVSK SEQ ID NO: 850 Q5F5N5 Pyruvate kinase GDLAVEVGHAAVPALQK SEQ ID NO: 851 Q5F5N5 Pyruvate kinase HNITLPIFALTPSVSAQR SEQ ID NO: 852 Q5F9F5 Ribose-phosphate FSDGEVAVELLENVR SEQ ID NO: 853 pyrophosphokinase Q5F9F5 Ribose-phosphate SVRVPISAK SEQ ID NO: 854 pyrophosphokinase Q5F9F5 Ribose-phosphate LVANMLYSAGIDR SEQ ID NO: 855 pyrophosphokinase Q5F9F5 Ribose-phosphate ANVAEVMNIIGDIQGK SEQ ID NO: 856 pyrophosphokinase Q5F9F5 Ribose-phosphate IASSEIDQVVVTDTIPLSEAAK SEQ ID NO: 857 pyrophosphokinase Q5F9F5 Ribose-phosphate QVTIAGLLAETVR SEQ ID NO: 858 pyrophosphokinase Q5F666 Signal recognition particle ILGMGDVLTLIEDVQK SEQ ID NO: 859 protein Q5F666 Signal recognition particle IAMGAGTTVQEVNK SEQ ID NO: 860 protein Q5F666 Signal recognition particle IAMGAGTTVQEVNKLLK SEQ ID NO: 861 protein Q5F7Y0 Single-stranded DNA- NENSGGAPYDEGYGQSQEAYQRPAQQSR SEQ ID NO: 862 binding protein Q5F7Y0 Single-stranded DNA- QPAPDAPSHPQEAPAAPR SEQ ID NO: 863 binding protein Q5F879 Succinyl-CoA ligase SVLINKDTK SEQ ID NO: 864 [ADP-forming] subunit alpha Q5F879 Succinyl-CoA ligase NGTFHSEQALAYGTK SEQ ID NO: 865 [ADP-forming] subunit alpha Q5F879 Succinyl-CoA ligase LVGPNCPGVITPGECK SEQ ID NO: 866 [ADP-forming] subunit alpha Q5F879 Succinyl-CoA ligase IGIMPGHIHTPGR SEQ ID NO: 867 [ADP-forming] subunit alpha Q5F879 Succinyl-CoA ligase MGHAGAIISGGK SEQ ID NO: 868 [ADP-forming] subunit alpha Q5F879 Succinyl-CoA ligase SPAELGTTMLEVLK SEQ ID NO: 869 [ADP-forming] subunit alpha Q5F5R0 Transcription ANRPTPISQR SEQ ID NO: 870 antitermination protein nusG Q5FA35 Transcription termination DLKSEENLTGR SEQ ID NO: 871 factor Rho Q5FA35 Transcription termination AYNTVVPASGK SEQ ID NO: 872 factor Rho Q5FA35 Transcription termination ILTGGVDANALHRPK SEQ ID NO: 873 factor Rho Q5F7X0 Tryptophan-tRNA ligase VLTGVTTTGTPHLGNYVGAIRPAVR SEQ ID NO: 874 Q5F7X0 Tryptophan-tRNA ligase YNALTSNPSQIEEILQAGAQK SEQ ID NO: 875 Q5F5L5 Twitching motility/pilus MQITDLLAFGAK SEQ ID NO: 876 retraction protein Q5F5L5 Twitching motility/pilus NKASDLHLSSGISPMIR SEQ ID NO: 877 retraction protein Q5F5L5 Twitching motility/pilus ASDLHLSSGISPMIR SEQ ID NO: 878 retraction protein Q5F5L5 Twitching motility/pilus FRVNAFNTGR SEQ ID NO: 879 retraction protein Q5F5L5 Twitching motility/pilus TIPSTVLSLEELKAPSIFQK SEQ ID NO: 880 retraction protein Q5F5L5 Twitching motility/pilus KSLINQR SEQ ID NO: 881 retraction protein Q5F5L5 Twitching motility/pilus SMLSESLTAVISQNLLK SEQ ID NO: 882 retraction protein Q5F5L5 Twitching motility/pilus VASHEILIANPAVR SEQ ID NO: 883 retraction protein

REFERENCES

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference in their entirety.

-   U.S. Patent Application Number 20080305157 -   Richard et. al., Clinical and Vaccine Immunology; 2014, 21(2):212 -   Alexander, J.; Del Guercio, M.-F.; Maewal, A.; Qiao, L.; Fikes, J.;     Chesnut, R. W.; Paulson, J.; Bundle, D. R.; DeFrees, S.; Sette, A.,     Linear PADRE T helper epitope and carbohydrate B cell epitope     conjugates induce specific high titer IgG antibody responses. J     Immunol. 2000, 164 (3), 1625-1633. -   Alnemri E S. 2010. Sensing cytoplasmic danger signals by the     inflammasome. J. Clin. Immunol. 30:512-519.     http://dx.doi.org/10.1007/s10875-010-9419-0. -   Alving, C. R., Lipopolysaccharide, lipid A, and liposomes containing     lipid A as immunologic adjuvants. Immunobiology 1993, 187 (3-5),     430-46. -   Alving, C. R.; Rao, M., Lipid A and liposomes containing lipid A as     antigens and adjuvants. Vaccine 2008, 26 (24), 3036-3045. -   Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M.,     N-Hydroxysuccinimide esters in peptide synthesis. J Am. Chern. Soc.     1963, 85 (19), 3039. -   Asare R, Kwaik Y A. 2010. Exploitation of host cell biology and     evasion of immunity by Francisella tularensis. Front. Microbiol.     1:145. http://dx.doi.org/10.3389/fmicb.2010.00145. -   Ashtekar A R, Katz J, Xu Q, Michalek S M. 2012. A mucosal subunit     vaccine protects against lethal respiratory infection with     Francisella tularensis LVS. PLoS One 7:e50460.     http://dx.doi.org/10.1371/journal.pone.0050460. -   Augustyniak, D.; Mleczko, J.; Gutowicz, J., The immunogenicity of     the liposome associated outer membrane proteins (OMPs) of Moraxella     catarrhalis. Cell Mol Biol Lett 2010, 15 (1), 70-89. -   Baron S D, Singh R, Metzger D W. 2007. Inactivated Francisella     tularensis live vaccine strain protects against respiratory     tularemia by intranasal vaccination in an immunoglobulin A-dependent     fashion. Infect. Immun. 75: 2152-2162.     http://dx.doi.org/10.1128/IAI.01606-06. -   Barratt, G., Colloidal drug carriers: Achievements and perspectives.     Cell. Mol. Life Sci. 2003, 60 (1), 21-37. -   Barrigan L M, Tuladhar S, Brunton J C, Woolard M D, Chen C J, Saini     D, Frothingham R, Sempowski G D, Kawula T H, Frelinger J A. 2013.     Infection with Francisella tularensis live vaccine strain clpB leads     to an altered yet protective immune response. Infect. Immun.     81:2028-2042. http://dx.doi.org/10.1128/IAI.00207-13. -   Barry E M, Cole L E, Santiago A E. 2009. Vaccines against tularemia.     Hum. Vaccin. 5:832-838. -   Bergsbaken T, Fink S L, Cookson B T. 2009. Pyroptosis: host cell     death and inflammation. Nat. Rev. Microbiol. 7:99-109.     http://dx.doi.org/10.1038/nrmicro2070. -   Bjune, G.; Hoiby, E. A.; Gronnesby, J. K.; Arnesen, O.;     Fredriksen, J. H.; Halstensen, A.; Holten, E.; Lindbak, A. K.;     Nokleby, H.; Rosenqvist, E.; et al., Effect of outer membrane     vesicle vaccine against group B meningococcal disease in Norway.     Lancet 1991, 338 (8775), 1093-6. -   Bouma, S. R.; Drislane, F. W.; Huestis, W. H., Selective extraction     of membrane bound proteins by phospholipid vesicles. J Biol Chem     1977, 252 (19), 6759-63. -   Cartwright, K.; Morris, R.; Rumke, H.; Fox, A.; Borrow, R.; Begg,     N.; Richmond, P.; Poolman, J., Immunogenicity and reactogenicity in     UK infants of a novel meningococcal vesicle vaccine containing     multiple class 1 (PorA) outer membrane proteins. Vaccine 1999, 17     (20-21), 2612-9. -   Celli J, Zahrt T C. 2013. Mechanisms of Francisella tularensis     intracellular pathogenesis. Cold Spring Harb. Perspect. Med.     3:a010314. http://dx.doi.org/10.1101/cshperspect.a010314. -   Chen W, KuoLee R, Shen H, Búsa M, Conlan J W. 2004. Toll-like     receptor 4 (TLR4) does not confer a resistance advantage on mice     against low-dose aerosol infection with virulent type A Francisella     tularensis. Microb. Pathog. 37:185-191.     http://dx.doi.org/10.1016/j.micpath.2004.06 0.010. -   Chen W, Shen H, Webb A, KuoLee R, Conlan J W. 2003. Tularemia in     BALB/c and C57BL/6 mice vaccinated with Francisella tularensis LVS     and challenged intradermally, or by aerosol with virulent isolates     of the pathogen: protection varies depending on pathogen virulence,     route of exposure, and host genetic background. Vaccine     21:3690-3700. http://dx.doi.org/10.1016/S0264-410X(03)00386-4. -   Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A.,     Multifunctional nanoparticles: Cost versus benefit of adding     targeting and imaging capabilities. Science 2012, 338 (6109),     903-910. -   Chevallet, M.; Luche, S.; Rabilloud, T., Silver staining of proteins     in polyacrylamide gels. Nat. Protoc. 2006, 1 (4), 1852-1858. -   Choksawangkam, W.; Kim, S.-K.; Cannon Joe, R.; Edwards Nathan, J.;     Lee Sang, B.; Fenselau, C., Enrichment of plasma membrane proteins     using nanoparticle pellicles: comparison between silica and higher     density nanoparticles. J Proteome Res 2013. -   Clay C D, Soni S, Gunn J S, Schlesinger L S. 2008. Evasion of     complement-mediated lysis and complement C3 deposition are regulated     by Francisella tularensis lipopolysaccharide O antigen. J. Immunol.     181: 5568-5578. -   Clemens D L, Lee B Y, Horwitz M A. 2004. Virulent and avirulent     strains of Francisella tularensis prevent acidification and     maturation of their phagosomes and escape into the cytoplasm in     human macrophages. Infect. Immun. 72:3204-3217.     http://dx.doi.org/10.1128/IAI.72.6.3204-3217.2004. -   Cole L E, Elkins K L, Michalek S M, Qureshi N, Eaton U, Rallabhandi     P, Cuesta N, Vogel S N. 2006. Immunologic consequences of     Francisella tularensis live vaccine strain infection: role of the     innate immune response in infection and immunity. J. Immunol.     176:6888-6899. -   Cole L E, Mann B J, Shirey K A, Richard K, Yang Y, Gearhart P J,     Chesko K L, Viscardi R M, Vogel S N. 2011. Role of TLR signaling in     Francisella tularensis-LPS-induced, antibody-mediated protection     against Francisella tularensis challenge. J. Leukoc. Biol.     90:787-797. http://dx.doi.org/10.1189/jlb.0111014. -   Cole L E, Santiago A, Barry E, Kang T J, Shirey K A, Roberts Z J,     Elkins K L, Cross A S, Vogel S N. 2008. Macrophage proinflammatory     response to Francisella tularensis live vaccine strain requires     coordination of multiple signaling pathways. J. Immunol.     180:6885-6891. -   Cole L E, Shirey K A, Barry E, Santiago A, Rallabhandi P, Elkins K     L, Puche A C, Michalek S M, Vogel S N. 2007. Toll-like receptor     2-mediated signaling requirements for Francisella tularensis live     vaccine strain infection of murine macrophages. Infect. Immun.     75:4127-4137. http://dx.doi.org/10.1128/IAI.01868-06. -   Cole L E, Yang Y, Elkins K L, Fernandez E T, Qureshi N, Shlomchik M     J, Herzenberg L A, Herzenberg L A, Vogel S N. 2009. Antigen-specific     B-1a antibodies induced by Francisella tularensis LPS provide     long-term protection against F. tularensis LVS challenge. Proc.     Natl. Acad. Sci. U.S.A. 106:4343-4348.     http://dx.doi.org/10.1073/pnas.0813411106. -   Conlan J W, Chen W, Bosio C M, Cowley S C, Elkins K L. 2011.     Infection of mice with Francisella as an immunological model. Curr.     Protoc. Immunol. Chapter 19:Unit 19.14.     http://dx.doi.org/10.1002/0471142735.im1914s93. -   Conlan J W, Shen H, Golovliov I, Zingmark C, Oyston P C, Chen W,     House R V, Sjöstedt A. 2010. Differential ability of novel     attenuated targeted deletion mutants of Francisella tularensis     subspecies tularensis strain SCHU S4 to protect mice against aerosol     challenge with virulent bacteria: effects of host background and     route of immunization. Vaccine 28:1824-1831.     http://dx.doi.org/10.1016/j.vaccine.2009.12.001. -   Corbel, M. J., Reasons for instability of bacterial vaccines. Dev.     Biol. Stand. -   1996, 87, 113-124. -   Cowley S C, Elkins K L. 2011. Immunity to Francisella. Front.     Microbiol. 2:26. http://dx.doi.org/10.3389/fmicb.2011.00026. -   Crane D D, Warner S L, Bosio C M. 2009. A novel role for plasmid     mediated degradation of opsonizing antibody in evasion of host     immunity by virulent, but not attenuated, Francisella tularensis. J.     Immunol. 183: 4593-4600. http://dx.doi.org/10.4049/jimmuno1.0901655. -   Cremer T J, Amer A, Tridandapani S, Butchar J P. 2009. Francisella     tularensis regulates autophagy-related host cell signaling pathways.     Autophagy 5:125-128. http://dx.doi.org/10.4161/auto.5.1.7305. -   Daghastanli Katia, R. P.; Ferreira Rinaldo, B.; Thedei, G., Jr.;     Maggio, B.; Ciancaglini, P., Lipid composition-dependent     incorporation of multiple membrane proteins into liposomes. Colloids     SuifB Biointeifaces 2004, 36 (3-4), 127-37. -   Danoff E J, Wang X, Tung S H, Sinkov N A, Kemme A M, Raghavan S R,     English D S. 2007. Surfactant vesicles for high-efficiency capture     and separation of charged organic solutes. Langmuir 23:8965-8971.     http://dx.doi.org/10.1021/1a070215n. -   de Moreno M R, Smith J F, Smith R V. 1985. Silver staining of     proteins in polyacrylamide gels: increased sensitivity through a     combined Coomassie blue-silver stain procedure. Anal. Biochem.     151:466-470. http://dx.doi.org/10.1016/0003-2697(85)90206-4. -   De Pascalis R, Chou A Y, Bosio C M, Huang C Y, Follmann D A, Elkins     K L. 2012. Development of functional and molecular correlates of     vaccine-induced protection for a model intracellular pathogen, F.     tularensis LVS. PLoS Pathog. 8:e1002494.     http://dx.doi.org/10.1371/journal.ppat.1002494. -   Denes A, Lopez-Castejon G, Brough D. 2012. Caspase-1: is IL-1 just     the tip of the ICEberg? Cell Death Dis. 3:e338.     http://dx.doi.org/10.1038/cddis.2012.86. -   Dijkstra, J.; Mellors, J. W.; Ryan, J. L., Altered in vivo activity     of liposome incorporated lipopolysaccharide and lipid A. Infect.     Immun. 1989, 57 (11), 3357-63. -   Dreisenbach V, Cowley S, Elkins K L. 2000. Purified     lipopolysaccharide from Francisella tularensis live vaccine strain     (LVS) induces protective immunity against LVS infection that     requires B cells and gamma interferon. Infect. Immun. 68:1988-1996.     http://dx.doi.org/10.1128/IAI.68.4.1988-1996.2000. -   Dueñas A I, Aceves M, Orduña A, Diaz R, Sánchez Crespo M,     García-Rodríguez C. 2006. Francisella tularensis LPS induces the     production of cytokines in human monocytes and signals via Toll-like     receptor 4 with much lower potency than E. coli LPS. Int. Immunol.     18:785-795. http://dx.doi.org/10.1093/intimm/dx1015. -   Eigelsbach H T, Braun W, Herring R D. 1951. Studies on the variation     of Bacterium tularense. J. Bacteriol. 61:557-569. -   Elkins K L, Colombini S M, Meierovics A I, Chu M C, Chou A Y, Cowley     S C. 2010. Survival of secondary lethal systemic Francisella LVS     challenge depends largely on interferon gamma Microbes Infect.     12:28-36. http://dx.doi.org/10.1016/j.micinf.2009.09.012. -   Elkins K L, Cowley S, BosioCM.2003. Innate and adaptive     immuneresponses to an intracellular bacterium, Francisella     tularensis live vaccine strain. Microbes Infect. 5:135-142.     http://dx.doi.org/10.1016/S1286-4579(02)00084-9. -   Ellis, T. N.; Kuehn, M. J., Virulence and immunomodulatory roles of     bacterial outer membrane vesicles. Microbial. Mol. Biol. Rev. 2010,     74 (1), 81-94. -   Faraji, A. H.; Wipf, P., Nanoparticles in cellular drug delivery.     Bioorg. Med. Chern. 2009, 17 (8), 2950-2962. 134 -   Foley J E, Nieto N C. 2010. Tularemia. Vet. Microbiol. 140:332-338.     http://dx.doi.org/10.1016/j.vetmic.2009.07.017. -   Forestal C A, Malik M, Catlett S V, Savitt A G, Benach J L, Sellati     T J, Furie M B. 2007. Francisella tularensis has a significant     extracellular phase in infected mice. J. Infect. Dis. 196:134-137.     http://dx.doi.org/10.1086/518611. -   Forssen, E. A.; Ross, M. E., Daunoxome treatment of solid tumors:     Preclinical and clinical investigations. J Liposome Res. 1994,     481-512. -   Foster, K. A.; Gorringe, A. R.; Hudson, M. J.; Reddin, K. M.;     Robinson, A. Preparation of outer membrane vesicles from Gram     negative bacteria and uses thereof as vaccines.     2002-GB57182003051379, 20021217, 2003. -   Girard, P.; Pecreaux, J.; Lenoir, G.; Faison, P.; Rigaud, J.-L.;     Bassereau, P., A new method for the reconstitution of membrane     proteins into giant unilamellar vesicles. Biophys. J 2004, 87 (1),     419-429. -   Golovliov I, Baranov V, Krocova Z, Kovarova H, Sjöstedt A. 2003. An     attenuated strain of the facultative intracellular bacterium     Francisella tularensis can escape the phagosome of monocytic cells.     Infect. Immun. 71:5940-5950.     http://dx.doi.org/10.1128/IAI.71.10.5940-5950.2003. -   Gregoriadis, G.; Leathwood, P. D.; Ryman, B. E., Enzyme entrapment     in liposomes. FEBS Letters 1971, 14 (2), 95-9. -   Gregory A E, Titball R, Williamson D. 2013. Vaccine delivery using     nanoparticles. Front. Microbiol. 3:13.     http://dx.doi.org/10.3389/fcimb.2013.00013. -   Gupta, R. K.; Siber, G. R., Adjuvants for human vaccines-current     status, problems and future prospects. Vaccine 1995, 13 (14),     1263-76. -   Hajjar A M, Harvey M D, Shaffer S A, Goodlett D R, Sjöstedt A,     Edebro H, Forsman M, Byström M, Pelletier M, Wilson C B, Miller S I,     Skerrett S J, Ernst R K. 2006. Lack of in vitro and in vivo     recognition of Francisella tularensis subspecies lipopolysaccharide     by Toll-like receptors. Infect. Immun. 74:6730-6738.     http://dx.doi.org/10.1128/IAI.00934-06. -   Harris J, Sharp F A, Lavelle E C. 2010. The role of inflammasomes in     the immunostimulatory effects of particulate vaccine adjuvants.     Eur. J. Immunol. 40:634-638.     http://dx.doi.org/10.1002/eji.200940172. -   Hartley G, Taylor R, Prior J, Newstead S, Hitchen P G, Morris H R,     Dell A, Titball R W. 2006. Grey variants of the live vaccine strain     of Francisella tularensis lack lipopolysaccharide O-antigen, show     reduced ability to survive in macrophages and do not induce     protective immunity in mice. Vaccine 24:989-996.     http://dx.doi.org/10.1016/j.vaccine.2005.08.075. -   Henry T, Brotcke A, Weiss D S, Thompson L J, Monack D M. 2007. Type     I interferon signaling is required for activation of the     inflammasome during Francisella infection. J. Exp. Med. 204:987-994.     http://dx.doi.org/10.1084/jem.20062665. -   Hickey A J, Hazlett K R, Kirimanjeswara G S, Metzger D W. 2011.     Identification of Francisella tularensis outer membrane protein A     (FopA) as a protective antigen for tularemia. Vaccine 29:6941-6947.     http://dx.doi.org/10.1016/j.vaccine.2011.07.075. -   Holst, J.; Martin, D.; Arnold, R.; Huergo, C. C.; Oster, P.;     O'Hallahan, J.; Rosenqvist, E., Properties and clinical performance     of vaccines containing outer membrane vesicles from Neisseria     meningitidis. Vaccine 2009, 27 (Suppl. 2), B3-B12. -   Hornick R B, Eigelsbach H T. 1966. Aerogenic immunization of man     with live Tularemia vaccine. Bacteriol. Rev. 30:532-538. -   Humphries, H. E.; Williams, J. N.; Blackstone, R.; Jolley, K. A.;     Yuen, H. M.; Christodoulides, M.; Heckels, J. E., Multivalent     liposome-based vaccines containing different serosubtypes of PorA     protein induce cross-protective bactericidal immune responses     against Neisseria meningitidis. Vaccine 2006, 24 (1), 36-44. -   Idanpaan-Heikkila, I.; Hoiby, E. A.; Chattopadhyay, P.; Airaksinen,     U.; Michaelsen, T. M.; Wedege, E., Antibodies to meningococcal class     1 outer-membrane protein and its variable regions in patients with     systemic meningococcal disease. J Med Microbiol 1995, 43 (5),     335-43. -   Jia Q, Lee B Y, Bowen R, Dillon B J, Som S M, Horwitz M A. 2010. A     Francisella tularensis live vaccine strain (LVS) mutant with a     deletion in capB, encoding a putative capsular biosynthesis protein,     is significantly more attenuated than LVS yet induces potent     protective immunity in mice against F. tularensis challenge. Infect.     Immun. 78:4341-4355. http://dx.doi.org/10.1128/IAL00192-10. -   Jones C L, Napier B A, Sampson T R, Llewellyn A C, Schroeder M R,     Weiss D S. 2012. Subversion of host recognition and defense systems     by Francisella spp. Microbiol. Mol. Biol. Rev. 76:383-404.     http://dx.doi.org/10.1128/MMBR.05027-11. -   Juruj C, Lelogeais V, Pierini R, Perret M, Py B F, Jamilloux Y, Broz     P, Ader F, Faure M, Henry T. 2013. Caspase-1 activity affects AIM2     speck formation/stability through a negative feedback loop. Front.     Cell. Infect. Microbiol. 3:14.     http://dx.doi.org/10.3389/fcimb.2013.00014. -   Kaler E W, Murthy A K, Rodriguez B E, Zasadzinski J A. 1989.     Spontaneous vesicle formation in aqueous mixtures of single-tailed     surfactants. Science 245:1371-1374.     http://dx.doi.org/10.1126/science.2781283. -   Katz J, Zhang P, Martin M, Vogel S N, Michalek S M. 2006. Toll-like     receptor 2 is required for inflammatory responses to Francisella     tularensis LVS. Infect. Immun. 74:2809-2816.     http://dx.doi.org/10.1128/IAI.74.5 0.2809-2816.2006. -   Kersten G F, Crommelin D J. 2003. Liposomes and ISCOMs. Vaccine     21:915-920. http://dx.doi.org/10.1016/S0264-410X(02)00540-6. -   Kieffer T L, Cowley S, Nano F E, Elkins K L. 2003. Francisella     novicida LPS has greater immunobiological activity in mice than F.     tularensis LPS, and contributes to F. novicida murine pathogenesis.     Microbes Infect. 5:397-403.     http://dx.doi.org/10.1016/S1286-4579(03)00052-2. -   Kingsley Jeffrey, D.; Dou, H.; Morehead, J.; Rabinow, B.; Gendelman     Howard, E.; Destache Christopher, J., Nanotechnology: a focus on     nanoparticles as a drug delivery system. J Neuroimmune Pharmacal.     2006, 1 (3), 340-50. -   Koskela P, Herva E. 1982. Cell-mediated and humoral immunity induced     by a live Francisella tularensis vaccine. Infect. Immun. 36:983-989. -   KuoLee R, Harris G, Conlan J W, Chen W. 2011. Role of neutrophils     and NADPH phagocyte oxidase in host defense against respiratory     infection with virulent Francisella tularensis in mice. Microbes     Infect. 13:447-456. http://dx.doi.org/10.1016/j.micinf.2011.01.010. -   Kurtz S L, Foreman O, Bosio C M, Anver M R, Elkins K L. 2013.     Interleukin-6 is essential for primary resistance to Francisella     tularensis live vaccine strain infection. Infect. Immun. 81:585-597.     http://dx.doi.org/10.1128/IAL01249-12. -   Lasic, D. D., Novel applications of liposomes. Trends Biotechnol.     1998, 16 (7), 307-321. -   Macdonald, A. G.; Martinac, B.; Bartlett, D. H., Patch-clamp     experiments with porins extracted from a marine bacterium (Photo     bacterium profundum strain SS9) and reconstituted in liposomes. Cell     Biochem. Biophys. 2002, 37 (3), 157-167. -   Mahawar M, Rabadi S M, Banik S, Catlett S V, Metzger D W, Malik M,     Bakshi C S. 2013. Identification of a live attenuated vaccine     candidate for tularemia prophylaxis. PLoS One 8:e61539.     http://dx.doi.org/10.1371/journal.pone.0061539. -   Mann B J, Ark N M. 2009. Rationally designed tularemia vaccines.     Expert Rev. Vaccines 8:877-885. http://dx.doi.org/10.1586/erv.09.51. -   Mara-Koosham G, Hutt J A, Lyons C R, Wu T H. 2011. Antibodies     contribute to effective vaccination against respiratory infection by     type A Francisella tularensis strains. Infect. Immun. 79:1770-1778.     http://dx.doi.org/10.1128/IAL00605-10. -   Mares C A, Ojeda S S, Morris E G, Li Q, Teale J M. 2008. Initial     delay in the immune response to Francisella tularensis is followed     by hypercytokinemia characteristic of severe sepsis and correlating     with upregulation and release of damage-associated molecular     patterns. Infect. Immun. 76: 3001-3010.     http://dx.doi.org/10.1128/IAI.00215-08. -   McCrumb F R, Jr. 1961. Aerosol infection of man with Pasteurella     tularensis. Bacteriol. Rev. 25:262-267. -   Morein B, Hu K F, Abusugra I. 2004. Current status and potential     application of ISCOMs in veterinary medicine. Adv. Drug Deliv. Rev.     56:1367-1382. http://dx.doi.org/10.1016/j.addr.2004.02.004. -   Nieves, W.; Asakrah, S.; Qazi, O.; Brown, K. A.; Kurtz, J.;     AuCoin, D. P.; McLachlan, J. B.; Roy, C. J.; Morici, L. A., A     naturally derived outer-membrane vesicle vaccine protects against     lethal pulmonary Burkholderia pseudomallei infection. Vaccine 2011,     29 (46), 8381-8389. -   Ojogun V A, Lehmler H J, Knutson B L. 2009. Cationic-anionic vesicle     templating from fluorocarbon/fluorocarbon and     hydrocarbon/fluorocarbon surfactants. J. Colloid Interface Sci.     338:82-91. http://dx.doi.org/10.1016/j.jcis.2009.06.022. -   Oyston P C, Sjostedt A, Titball R W. 2004. Tularaemia: bioterrorism     defense renews interest in Francisella tularensis. Nat. Rev.     Microbiol. 2:967-978. http://dx.doi.org/10.1038/nrmicro1045. -   Pace, D.; Cuschieri, P.; Galea Debono, A.; Attard-Montalto, S.,     Epidemiology of pathogenic Neisseria meningitidis serogroup B     serosubtypes in Malta: Implications for introducing PorA based     vaccines. Vaccine 2008, 26 (47), 5952-5956. -   Park, J.-H.; Rader, L. H.; Thomas, G. B.; Danoff, E. J.; English, D.     S.; DeShong, P., Carbohydrate-Functionalized Surfactant Vesicles:     Preparation and Lectin-Binding Studies. Soft Matter 2008, Submitted. -   Parmar, M. M.; Edwards, K.; Madden, T. D., Incorporation of     bacterial membrane proteins into liposomes: factors influencing     protein reconstitution. Biochim. Biophys. Acta, Biomembr. 1999, 1421     (1), 77-90. -   Petrosino J F, Xiang Q, Karpathy S E, Jiang H, Yerrapragada S, Liu     Y, Gioia J, Hemphill L, Gonzalez A, Raghavan T M, Uzman A, Fox G E,     Highlander S, Reichard M, Morton R J, Clinkenbeard K D, Weinstock     G M. 2006. Chromosome rearrangement and diversification of     Francisella tularensis revealed by the type B (OSU18) genome     sequence. J. Bacteriol. 188:6977-6985.     http://dx.doi.org/10.1128/JB.00506-06. -   Prior J L, Prior R G, Hitchen P G, Diaper H, Griffin K F, Morris H     R, Dell A, Titball R W. 2003. Characterization of the O antigen gene     cluster and structural analysis of the O antigen of Francisella     tularensis subsp. tularensis. J. Med. Microbiol. 52:845-851.     http://dx.doi.org/10.1099/jmm.0.05184-0. -   Racker, E., Reconstitution of membrane processes. Methods Enzymol.     1979, 55 (Biomembranes, Part F), 699-711. -   Racker, E., Reconstitutions: past, present and future. Membr.     Bioenerg., Int. Workshop 1979, 569-91. -   Ramsey, R. B.; Hamner, M. B.; Alving, B. M.; Finlayson, J. S.;     Alving, C. R.; Evatt, B. L., Effects of lipid A and liposomes     containing lipid A on platelet and fibrinogen production in rabbits.     Blood 1980, 56 (2), 307-10. -   Richards, R. L.; Alving, C. R.; Wassef, N. M., Liposomal Subunit     Vaccines: Effects of Lipid A and Aluminum Hydroxide on     Immunogenicity. J Pharm. Sci. 1996, 85 (12), 1286-1289. -   Richards, R. L.; Rao, M.; Wassef, N. M.; Glenn, G. M.; Rothwell, S.     W.; Alving, C. R., Liposomes containing lipid A serve as an adjuvant     for induction of antibody and cytotoxic T-cell responses against     RTS,S malaria antigen. Infect. Immun. 1998, 66 (6), 2859-2865. -   Richards, R. L.; Swartz, G. M., Jr.; Schultz, C.; Hayre, M. D.;     Ward, G. S.; Ballou, W. R.; Chulay, J. D.; Hockmeyer, W. T.;     Berman, S. L.; Alving, C. R., Immunogenicity of liposomal malaria     sporozoite antigen in monkeys: adjuvant effects of aluminium     hydroxide and non-pyrogenic liposomal lipid A. Vaccine 1989, 7 (6),     506-12. -   Rigaud, J. L., Membrane proteins: functional and structural studies     using reconstituted proteoliposomes and 2-D crystals. Braz. J Med.     Biol. Res. 2002, 35 (7), 753-766. -   Rigaud, J. L.; Pitard, B., Liposomes as tools for the reconstitution     of biological systems. Liposomes Tools Basic Res. Ind. 1995, 71-88. -   Robbins, J. B.; Schneerson, R., Polysaccharide-protein conjugates: a     new generation of vaccines. J Infect. Dis. 1990, 161 (5), 821-32. -   Robinson D S, O'Garra A. 2002. Further checkpoints in Th1     development. Immunity 16:755-758.     http://dx.doi.org/10.1016/S1074-7613(02) 00331-X. -   Rockx-Brouwer D, Chong A, Wehrly T D, Child R, Crane D D, Celli J,     Bosio C M. 2012. Low dose vaccination with attenuated Francisella     tularensis strain SchuS4 mutants protects against tularemia     independent of the route of vaccination. PLoS One 7:e37752.     http://dx.doi.org/10.1371/journal.pone.0037752. -   Rothman, E. S.; Serota, S.; Swem, D., Enol esters. II. N-Acylation     of amides and imides. J Org. Chem. 1964, 29 (3), 646-50. -   Rubálek M, Hernychová L, Havlasová J, Kasalová I, Neubauerová V,     Stulík J, Macela A, Lundqvist M, Larsson P. 2003. Towards proteome     database of Francisella tularensis. J. Chromatogr. B Analyt.     Technol. Biomed. Life Sci. 787:149-177.     http://dx.doi.org/10.1016/S1570-0232(02)00730-4. -   Ryden P, Twine S, Shen H, Harris G, Chen W, Sjostedt A,     Conlan W. 2013. Correlates of protection following vaccination of     mice with gene deletion mutants of Francisella tularensis subspecies     tularensis strain, SCHU S4 that elicit varying degrees of immunity     to systemic and respiratory challenge with wild-type bacteria. Mol.     Immunol. 54:58-67. http://dx.doi.org/10.1016/j.molimm.2012.10.043. -   Samad, A.; Sultana, Y.; Aqil, M., Liposomal drug delivery systems:     an update review. Curr. Drug Deliv. 2007, 4 (4), 297-305. -   Sanchez, S.; Abel, A.; Marzoa, J.; Gorringe, A.; Criado, T.;     Ferreiros, C. M., Characterisation and immune responses to     meningococcal recombinant porin complexes incorporated into     liposomes. Vaccine 2009, 27 (39), 5338-5343. -   Šegota S, Težak D. 2006. Spontaneous formation of vesicles. Adv.     Colloid Interface Sci. 121:51-75.     http://dx.doi.org/10.1016/j.cis.2006.01.002. -   Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M.,     In-gel digestion for mass spectrometric characterization of proteins     and proteomes. Nat. Pro toe. 2006, 1 (6), 2856-2860. -   Shirey K A, Cole L E, Keegan A D, Vogel S N. 2008. Francisella     tularensis live vaccine strain induces macrophage alternative     activation as a survival mechanism. J. Immunol. 181:4159-4167. -   Silvius, J. R., Solubilization and functional reconstitution of     biomembrane components. Annu. Rev. Biophys. Biomol. Struct. 1992,21,     323-48. -   Singh A, Rahman T, Malik M, Hickey A J, Leifer C A, Hazlett K R,     Sellati T J. 2013. Discordant results obtained with Francisella     tularensis during in vitro and in vivo immunological studies are     attributable to compromised bacterial structural integrity. PLoS One     8:e58513. http://dx.doi.org/10.1371/journal.pone.0058513. -   Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.;     Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.;     Olson, B. J.; Klenk, D. C., Measurement of protein using     bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76-85. -   Stallforth, P.; Lepenies, B.; Adibekian, A.; Seeberger Peter, H.,     Carbohydrates: A frontier in medicinal chemistry. J Med. Chem. 2009,     52 (18), 5561-77. -   Stein, D. C.; Patrone, J. B.; Bish, S., Innate immune recognition of     Neisseria meningitidis and Neisseria gonorrhoeae. Neisseria 2010,     95-122. -   Tamauchi, H.; Tadakuma, T.; Yasuda, T.; Tsumita, T.; Saito, K.,     Enhancement of immunogenicity by incorporation of lipid A into     liposomal model membranes and its application to membrane-associated     antigens. Immunology 1983, 50 (4), 605-12. -   Thomas G B, Rader L H, Park J, Abezgauz L, Danino D, DeShong P,     English D S. 2009. Carbohydrate modified catanionic vesicles:     probing multivalent binding at the bilayer interface. J. Am. Chem.     Soc. 131:5471-5477. http://dx.doi.org/10.1021/ja8076439. -   Titball R W, Petrosino J F. 2007. Francisella tularensis genomics     and proteomics. Ann. N. Y. Acad. Sci. 1105:98-121.     http://dx.doi.org/10.1196/annals.1409.015. -   Torchilin, V. P., Recent advances with liposomes as pharmaceutical     carriers. Nat. Rev. Drug Disc. 2005, 4 (2), 145-160. -   Twine S, Shen H, Harris G, Chen W, Sjostedt A, Ryden P,     Conlan W. 2012. BALB/c mice, but not C57BL/6 mice immunized with a     _clpB mutant of Francisella tularensis subspecies tularensis are     protected against respiratory challenge with wild-type bacteria:     association of protection with post-vaccination and post-challenge     immune responses. Vaccine 30: 3634-3645.     http://dx.doi.org/10.1016/j.vaccine.2012.03.036. -   Vamier, A.; Kermarrec, F.; Blesneac, I.; Moreau, C.; Liguori, L.;     Lenormand, J. L.; Picollet-D'hahan, N., A Simple Method for the     Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles.     J Membr. Biol. 2010, 233 (1-3), 85-92. -   van de Waterbeemd, B.; Streefland, M.; van der Ley, P.; Zomer, B.;     van Dijken, H.; Martens, D.; Wijffels, R.; van der Pol, L., Improved     OMV vaccine against Neisseria meningitidis using genetically     engineered strains and a detergent-free purification process.     Vaccine 2010, 28 (30), 4810-4816. -   Varki, A., Biological roles of oligosaccharides: All of the theories     are correct. Glycobiology 1993, 3 (2), 97-130. -   Vermont, C. L.; van Dijken, H. H.; Kuipers, A. J.; van Limpt, C. J.     P.; Keijzers, W. C. M.; van der Ende, A.; de Groot, R.; van Alphen,     L.; van den Dobbelsteen, G. P. J. M., Cross-reactivity of antibodies     against PorA after vaccination with a meningococcal B outer membrane     vesicle vaccine. Infect. Immun. 2003, 71 (4), 1650-1655. -   Wang X, Danoff E J, Sinkov N A, Lee J H, Raghavan S R, English     D S. 2006. Highly efficient capture and long-term encapsulation of     dye by catanionic surfactant vesicles. Langmuir 22:6461-6464.     http://dx.doi.org/10.1021/1a0605135. -   Wang, X.; Quinn, P. J., Endotoxins: lipopolysaccharides of     gram-negative bacteria. Subcell. Biochem. 2010, 53 (Endotoxins),     3-25. -   Wassef, N. M.; Alving, C. R.; Richards, R. L., Liposomes as carriers     for vaccines. ImmunoMethods 1994, 4 (3), 217-22. -   Wayne Conlan J, Oyston P C. 2007. Vaccines against Francisella     tularensis. Ann. N. Y. Acad. Sci. 1105:325-350.     http://dx.doi.org/10.1196/annals.1409.012. -   Westphal, 0.; Jann, K.; Himmelspach, K., Chemistry and     immunochemistry of bacteriallipopolysaccharides as cell wall     antigens and endotoxins. Prog Allergy 1983, 33, 9-39. -   Wetzler, L. M.; Blake, M. S.; Barry, K.; Gotschlich, E. C.,     Gonococcal porin vaccine evaluation: comparison of Por proteosomes,     liposomes, and blebs isolated from rmp deletion mutants. J Infect     Dis 1992, 166 (3), 551-5. -   Wilton J M. 1978. Suppression by IgA of IgG-mediated phagocytosis by     human polymorphonuclear leucocytes. Clin. Exp. Immunol. 34:423-428. -   Working, P. K.; Newman, M. S.; Huang, S. K.; Mayhew, E.; Vaage, J.;     Lasic, D. D., Pharmacokinetics, biodistribution and therapeutic     efficacy of doxorubicin encapsulated in Stealth liposomes (Doxil). J     Liposome Res. 1994, 667-87. -   Wu T H, Hutt J A, Garrison K A, Berliba L S, Zhou Y, Lyons     C R. 2005. Intranasal vaccination induces protective immunity     against intranasal infection with virulent Francisella tularensis     biovar A. Infect. Immun. 73: 2644-2654.     http://dx.doi.org/10.1128/1A1.73.5.2644-2654.2005. -   Yamasaki, R.; Bacon, B. E.; Nasholds, W.; Schneider, H.;     Griffiss, J. M., Structural determination of oligosaccharides     derived from lipooligosaccharide of Neisseria gonorrhoeae F62 by     chemical, enzymatic, and two-dimensional NMR methods. Biochemistry     1991, 30 (43), 10566-75. -   Zakirov, M. M.; Petrov, A. B.; Burkhanov, S. A.; Vartanian Iu, P.;     Torchilin, V. P.; Trubetskoi, V. S.; Koshkina, N. V.; Dmitriev, B.     A.; L'Vov V, L., The immunological activity of Neisseria     meningitidis lipo-oligosaccharide incorporated into liposomes. Zh     Mikrobiol Epidemiol Immunobiol 1995, (1), 49-53. -   Zhu, J.; Xue, J.; Guo, Z.; Zhang, L.; Marchant, R. E., Biomimetic     glycoliposomes as nanocarners for targeting P-selectin on activated     platelets. Bioconjugate Chern. 2007, 18 (5), 1366-1369. -   Zhu, J.; Yan, F.; Guo, Z.; Marchant, R. E., Surface modification of     liposomes by saccharides: Vesicle size and stability of lactosyl     liposomes studied by photon correlation spectroscopy. J Colloid     Inteiface Sci. 2005, 289 (2), 542-550. -   Zollinger Wendell, D.; Babcock Janiine, G.; Moran Elizabeth, E.;     Brandt Brenda, L.; Matyas Gary, R.; WassefNabila, M.; Alving Carl,     R., Phase I study of a Neisseria meningitidis liposomal vaccine     containing purified outer membrane proteins and detoxified     lipooligosaccharide. Vaccine 2012, 30 (4), 712-21.

Example 1 Extraction of Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles

In this paper we describe how catanionic surfactant vesicles were used to extract membrane components from the Gram negative pathogen Neisseria gonorrhoeae. Catanionic vesicle extracts were prepared using the surfactants SDBS and CTAT in the presence of N. gonorrhoeae cell pellets. Colorimetric carbohydrate and protein assays of the resulting catanionic vesicles confirmed the presence of carbohydrate and protein in the resulting vesicles. Gel electrophoresis followed by silver staining further confirmed that vesicle extracts contained pathogen-derived LOS F62ΔlgtD and a subset of proteins. Western blotting confirmed that a select number of proteins were from the outer membrane of the N. gonorrhoeae bacterium, including porin and OPA. Mass spectrometric analysis of the catanionic vesicle extracts identified 138 total proteins, 29 of which were membrane proteins.

Methods

All chemicals and solvents were purchased from commercial suppliers and were used as received unless otherwise noted. All aqueous vesicle solutions were prepared from a Millipore (18MQ) water purification system and all assays used water purified using an Elix 5 (Millipore) water purification system unless otherwise stated. An Ocean Optics USB 2000 Spectrometer was used to measure UV-VIS absorbance of samples.

Cell Cultures.

Genetically modified N gonorrhoeae F62˜lgtD cells were grown to a maximum cell count for 48 h with an OD of 0.6-1.O (650 nm) (106-109 CFU). A 20 mL aliquot of bacterial cell culture was spun down by centrifugation at 9,000 RPM for 30 min and the supernatant was decanted. Cell pellets were stored at −20° C. until needed.

Vesicle Preparation.

Sodium dodecylbenzenesulfonate (SDBS) was purchased from TCI America and was utilized without further purification. Cetyltrimethylammonium tosylate (CTAT) was purchased from Sigma and was recrystallized from ethanol-acetone to give a white powder. The purified solid was stored at room temperature in a desiccator containing Drierite.

Several vesicle preparation methods were tested to determine if the extraction varied between the order and type of addition of the surfactant components. The decided upon method, described below, was chosen based on Western blotting, which yielded the most protein bands. Procedures for the other preparation methods can be found in the Supporting Information.

Vesicle extracts were formed by adding a solution of SDBS directly to the bacterial cell pellet then adding solid CTAT to form 1 wt % total surfactant (26.9 mM total surfactant). This was done by adding 9.90 mL of an aqueous SDBS solution (0.0203 M) directly to the bacterial cell pellet and stirring for 1 hour at room temperature. Then 30.0 mg of solid CTAT (0.0658 mmol) was added to the suspension and stirred for 1 hour at room temperature.

Vesicles were centrifuged for 5 min at 5,000 RPM and the supernatant was decanted. The resulting colloidal supernatant, milky in appearance, was purified by gel filtration on Sephadex G-100 where a 1.0 mL aliquot of vesicle extract solution was added to a column (length 5.5 cm, diameter 1.5 cm) packed with G-100 Sephadex (Sigma). Vesicles were eluted with 1.0 mL aliquots of water to a total volume of 14.0 mL, yielding fourteen fractions.

Characterization of Vesicles.

All fractions from gel filtration were characterized for the presence of carbohydrate using a phenol-sulfuric colorimetric assay (FIG. 1). 41 Absorption studies by UV-VIS at 490 nm indicated the presence of carbohydrate in vesicle-containing fractions.

Vesicles were also analyzed for the presence of protein using a modified procedure of the Pierce bicinchoninic acid (BCA) assay (FIG. 1).42 The working reagent was prepared using a 50:1 v/v ratio of Reagent A (sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartrate in 0.1 M sodium hydroxide) to Reagent B (4% copper (II) sulfate). The test-tube protocol was used in which 2.0 mL of working reagent was added to 0.1 mL of the sample. To prevent intact vesicles from scattering light and interfering with the absorbance of samples, 0.1 mL of 1-propanol was added to each sample to break up vesicles. After the addition of the working reagent, the samples were vortexed and incubated at 37° C. for 30 min. The absorbance was measured at 562 nm and compared to a bovine serum albumin standard curve to determine the total protein concentration in each sample.

Gel Electrophoresis.

Vesicle samples and a molecular weight standard were mixed with loading buffer (3×,details) and boiled for 10 min. Samples were loaded onto an SDS-polyacrylamide gel (Tris-tricine 16.5% v/v) using Tris-tricine IX as the running buffer and run for 4.5 h at 100 Von ice (Bio-Rad Model 200/2.0 power supply).

Silver Staining.

After electrophoresis, the gels were incubated in a fixing solution (500 mL of 38% ethanol and 25 mL glacial acetic acid) overnight on a shaker at room temperature. Gels were silver stained according to a modified procedure. 43 The gel was transferred to 100 mL of an aqueous periodic acid (0.036 M) wash for 5 min and then rinsed four times with water for 30 min on a shaker. The silver staining solution was prepared by adding 4.0 mL of diluted silver nitrate (4.7 mmol) drop wise to Solution 1 (1 pellet sodium hydroxide, 25 mL water, 1.40 mL of 30% ammonium hydroxide). If a brown color was present, additional ammonium hydroxide was added drop wise until the solution became clear and colorless. The silver staining solution was brought to a final volume of 100 mL with water and the gel was incubated for 15 min with the solution on a shaker at room temperature.

After silver staining, the gel was washed with water six times for 15 min each. The gel was then incubated in a developing solution (95 μL formaldehyde 37% solution, 1 mL citric acid 25 mg/mL, 500 mL water) until bands became visible. The gel was washed in water and then imaged.

Protection Experiments.

Vesicle-containing fractions were digested using 10 μL proteinase K (25 mg/mL) for a 500 μL sample incubated at 37° C. Aliquots were taken after 18 h, 23 h, and 46 h. A control of the cell pellet was prepared by suspending the pellet in 1.0 mL of water. Proteinase K was added to the cell pellet suspension and a sample of whole cell lysate and incubated at 37° C. Aliquots were taken after 18 h, 23 h, and 46 hand all samples were analyzed by electrophoresis and silver staining.

The original samples from the proteinase K digestion were also digested with trypsin (0.25%, Coming cellgro), where 10 μL was added to 500 μL of sample and incubated at 37° C. Aliquots were taken after 18 h, 23 h, and 46 hand all samples were analyzed by electrophoresis and silver staining.

Westing Blotting.

After electrophoresis, Western blotting was performed in which the gel was sandwiched with nitrocellulose and transferred for 30 min at 2 Amps in a Tris-tricine 1×/MeOH solution on ice. The nitrocellulose was then air dried for 10 min and incubated in casein filler solution on a shaker overnight at room temperature.

The nitrocellulose was rinsed with a PBS/Tween-20 solution 5 times for 15 min and then incubated with heat goat serum (1:500) primary antibody solution prepared in casein filler on a shaker for 2 h. The nitrocellulose was rinsed with a PBS 1×/Tween-20 solution five times for 15 min each and then incubated with donkey anti-goat HRP (Jackson ImmunoResearch laboratories Inc.) (1:100,000) secondary antibody solution prepared in casein filler on a shaker for 2 h. The nitrocellulose was rinsed with a PBS 1×/Tween-20 solution five times for 15 min each and then incubated with a standard Western blotting chemiluminescence solution (PerkinElmer, Waltham, Mass.) and analyzed using autoradiograph film.

Proteomics Analysis.

Vesicle extracts were prepared from cell pellets formed from 20 mL, 40 mL, or 60 mL of cells. Known amounts of protein were spotted from these preparations in each lane of a one-dimensional gel (Tris-HCl, 8-16% gradient). Whole cell lysate was spotted in a fourth lane. The gel was developed and stained with Coomassie blue stain (Sigma). Fifteen slices were cut from each lane and subjected to overnight in-gel tryptic digestion (13 ng/μL) using a standard procedure. 44 The resulting peptides were extracted and injected into a capLC-MS/MS LTQ-orbitrap (ThermoFisher, San Jose, Calif.) as described elsewhere. 45 Peptide and protein candidates were analyzed using the search program MASCOT 2.3 (Matrix Science, London, UK), and protein identifications were based on the number of associated tryptic peptides and the reliability of the peptide identifications. Two protein databases were searched, one compiled of all Neisseria sequences from NCBinr (www.ncbi.nlm.nih.gov.com) and one comprised from only the proteins in NCBinr from the species N. gonorrhoeae. Subcellular locations of the proteins were assigned using the Protein Information Resource.

Results

Vesicle extractions of N. gonorrhoeae were characterized using a colorimetric carbohydrate assay, a BCA protein assay, silver staining after gel electrophoresis, Western blotting, and mass spectrometry-based proteomics. The results indicate that membrane components from bacteria were extracted successfully into surfactant vesicles. (see Table 1). The best method for extraction was using an SDBS solution to disrupt the cell pellet followed by addition of solid CTAT to the lysate solution. We propose that this method worked most effectively because the solution of SDBS first lysed the bacteria, freeing surface antigens, and then solubilized all membrane components. After the addition of CTAT, membrane components were efficiently captured during the formation of vesicles and were able to readily associate with the vesicle bilayer.

All vesicle preparation methods were purified by gel filtration and all resulting fractions were tested for the presence of carbohydrate and protein using a carbohydrate colorimetric assay and the BCA protein assay (FIG. 1). Vesicles extracted by all five methods had comparable amounts of carbohydrate and protein. Both assays serve to quantitatively determine the amount of carbohydrate and protein, respectively.

Samples from loaded vesicles were compared to the whole cell lysate and original cell pellet (FIG. 2). The presence of LOS in vesicles was confirmed by electrophoresis of a standard of LOS F62ΔlgtD. Silver staining confirmed the presence of carbohydrate, specifically LOS F62ΔlgtD, seen at the bottom of the gel (FIG. 2). LOS is presumed to incorporate easily into vesicles because the lipid tail associates with the lipophilic bilayer. Different proteins were shown to be extracted using vesicles, compared to the lysate. Furthermore, samples extracted by vesicles had different protein profiles when compared to that of the original cell pellet. (FIG. 2). Gel electrophoresis followed by silver staining was performed on all fractions of the described method in order to characterize the carbohydrates and proteins that were incorporated into the vesicles (FIG. 3). These images indicate that numerous proteins were incorporated into the vesicles and few components come out in later fractions.

Western blotting of the loaded materials from these surfactant vesicles showed the presence of LOS and the membrane proteins porin (36 kD) and OPA (25-30 kD) (FIG. 5). Further analysis by mass spectrometry confirmed the presence of these proteins (FIG. 4). Analysis of tryptic digests of the lanes from vesicle-containing fractions by mass spectrometry also identified 293 unique proteins in the vesicle extract, including ribosomal proteins as well as outer membrane bound proteins.

Protection experiments were carried out to test if bacterial proteins are translocated to the interior of the vesicles. Loaded vesicles were digested with proteinase K to determine if being associated with vesicles protected proteins. A whole cell lysate was incubated as a control. Both vesicle samples and the whole cell lysate showed complete digestion using this enzyme (FIG. 6). Since proteinase K is a nonspecific digestion enzyme, these results demonstrate that any protein at the surface of vesicles was digested completely by the enzyme. This proteolytic enzyme does not digest the carbohydrate LOS.

Digestion of the loaded vesicles using trypsin gave a different pattern. Digested proteins in the form of peptide units were seen in great concentrations at the bottom of the gel (FIG. 7). The vesicle samples showed protection for a few proteins, while the whole cell lysate fraction was completely cleaved by trypsin. This indicates that vesicles contain proteins embedded in the bilayer, where they are protected from cleavage from trypsin, but not proteinase K.

In another proteomic experiment, material was extracted from three different starting amounts of cells. Four lanes were developed in the gel, containing 20 μg of whole cell lysate, 39 μg of sample from a 20 mL pellet, 52 μg of sample from a 40 mL pellet, and 67 μg of sample from a 60 mL pellet. After trypsin digestion, the peptides were analyzed by HPLC-MS/MS. A search against all proteins from the genus Neisseria identified 229 proteins with high reliability (FIG. 4). Of these, 45 were classified as membrane proteins. When the limited protein database was searched, which contained proteins associated only with the species N gonorrhoeae, 29 membrane proteins were identified out of 138 total identifications (FIG. 4). Major protein bands based on silver staining were found to contain outer membrane proteins. Western Blotting confirmed the presence of pilin, porin, and OPA.

Catanionic surfactant vesicles are soft, cell-like models that can incorporate antigens, making them ideal vaccine agents. This work offers a way to extract membrane components from pathogenic bacteria for potential vaccine formulation. Membrane components of a cell would be useful if they could be easily separated. The vesicle extraction procedure introduced here offers potential enrichment of LOS and other membrane components from N. gonorrhoeae. These vesicles are stable at room temperature for prolonged periods of time and offer an improvement over conventional liposomes. The described vesicle extraction is currently being studied for use with other pathogens. Future research will be devoted to challenging animals and testing to determine if protection occurred from vesicle extracts containing neisserial proteins. Because the structure of LOS involved in the meningococcal disease is identical to that expressed by the N. gonorrhoeae F62ΔlgtD used in vesicle extractions, additional work will be devoted to generating a universal vaccine able to protect against all serotypes of N. meningitidis.

Example 2 Loading Complex Carbohydrates and Peptides into Catanionic Surfactant Vesicles for Use in Vaccine Applications

We were interested in developing carbohydrate-based vaccines against Gramnegative bacteria, specifically N. gonorrhoeae and F. tularensis by utilizing catanionic surfactant vesicles for incorporation of surface antigens onto the outer leaflet. The resulting functionalized vesicles would display the antigens on the surface of the vesicle in a manner analogous to their presence in bacterial pathogens. Vaccine studies by our lab were performed using catanionic surfactant vesicles that contained bacterial liposaccharides (i.e. LOS and LPS) and Pan DR helper T cell epitope (PADRE) peptide conjugate. The peptide PADRE was chosen for co-incorporation into vesicles because the peptide is a synthetic epitope that has been shown to stimulate the production of IgG antibodies. Specifically, PADRE has been shown to augment the potency of vaccines designed to stimulate T-cells in developing a potent immune response against the carbohydrate antigens that could be presented. We undertook this study to determine if liposaccharides and PADRE inserted into a catanionic vesicles would be capable of eliciting an immune response against the carbohydrate component (FIG. 9).

Results

Catanionic Vaccines for Neisseria gonorrhoeae.

Surface antigens from Gram-negative bacteria trigger the immune response and therefore offer the potential to be used in vaccine development. Components on the N gonorrhoeae cell membrane that trigger immune stimulation are LOS, porins (PorA and PorB), pili, and OP A. To date, no one has been able to exploit the immunological potential of neisserial LOS as a vaccine candidate.

Previous research in the DeShong group utilized LOS from N. gonorrhoeae for its incorporation into catanionic vesicles. In order to avoid toxicity associated with lipid A, the lipid A region was cleaved (FIG. 10). A C12-conjugate was added to the oligosaccharide to provide an anchor for insertion into vesicles. While this method was effective when preparing functionalized vesicles, incorporation of the lipid A region into liposomes is known to lower its toxicity. Consequently, we studied vesicles loaded with the entire liposaccharide unit to determine if lipid A toxicity is removed after incorporation into catanionic systems.

Catanionic vesicles were prepared by adding native LOS derived from N. gonorrhoeae F62˜lgtD (a strain that produces lacto-N-neotetraose LOS) (FIG. 11). Addition of this isolated pathogenic component should decorate the exterior membrane of vesicles (FIG. 9). Vesicles were purified by SEC and the presence of saccharide in the vesicle-containing fractions was confirmed using the phenol/sulfuric acid assay (described previously) (FIG. 14). Next, we developed a glycoconjugate-based vaccine (TRIAD) that contained the liposaccharide component and an epitope unit that would trigger the immune system. We utilized LOS and Pan DR helper T cell epitope (PADRE) peptide conjugate that possesses the ability to bind to a large number of HLA class II molecules. PADRE (FIG. 12) was coupled with N-dodecanoylsuccinimide to form dodecanoic acid tethered PADRE conjugate that inserted into the vesicle bilayer (FIG. 13). Both components were inserted into surfactant vesicles using a 10:1 w/w of LOS and C12—PADRE, respectively, and were purified by SEC (FIG. 15). Vesicle-containing fractions were analyzed by the phenol/sulfuric acid carbohydrate assay and by fluorescence. Results confirmed the presence of carbohydrate and phenylalanine and tryptophan residues in vesicle-containing fractions from SEC. These catanionic surfactant vesicle formulations were stable at room temperature for years, unlike typical liposomal vaccine formulations. Furthermore, TRIAD is so robust that it can be autoclaved without any appreciable loss of structural integrity.

Animal Studies with Vesicle Antigens.

Mice were treated with TRIAD vaccine that contained LOS and C12-PADRE at a ratio of 10:1. Antibody levels were determined by ELISA immunoassay by immunizing with 2 mg of LOS equivalent. Antibody titers for both LOS and LOS/C12-PADRE vesicles showed the generation of antibodies after mice were inoculated. Treatment with LOS vesicles showed that only primary antibodies were present (IgM). These results indicated that only a primary immune response was achieved. Inoculation with LOS/C12-PADRE showed that our vaccine induced a high titer anti-LOS antibody response, with the majority of the elicited antibody being IgG (FIG. 16). Intraperitoneal immunization of mice with our vaccine construct produced no observable adverse effects in mice, while intraperitoneal immunization with equivalent amounts of purified LOS induced significant adverse effects. Therefore, catanionic vesicles loaded with LOS/C12-PADRE may have generated IgG antibodies that recognize LOS (FIG. 9). Immunogenic recognition of LOS in our TRIAD carbohydrate-based vaccine and may offer protection against N. gonorrhoeae in challenged mice. Furthermore, the LOS from the strain F62˜lgtD contains the same LOS found in N. meningitidis. Therefore, a vaccine derived from F62˜lgtD LOS could offer protection against both gonorrhea and meningitis. This would allow a method to make vaccines against both pathogens without having to work directly with N. meningitidis.

Catanionic Vaccines for Francisella tularensis.

Similar catanionic vaccines were performed in F. tularensia. Catanionic vesicles were prepared in the same manner as before, but native LPS from F. tularensis was substituted for LOS (FIG. 17). After inoculation of LPS control vesicles, mice challenged with the virulent strain of F. tularensis died within four hours. Conversely, four out of five mice inoculated with LPS/C12-PADRE vesicles survived for four days after being challenged. One mouse even survived for two weeks. These results showed that mice vaccinated using LPS/C12-PADRE vesicles survived longer than the control group. These vaccines offer promise toward the development of vesicle-based vaccines to combat tularemia infections. Further vaccine studies are currently underway to determine the antibody titers using the ELISA immunoassay. Furthermore, the ratio of peptide conjugate to saccharide can be controlled in vesicle-based vaccines in order to control the immune system's response to the peptide.

We have devised a safe and effective way of generating a large IgG titer against carbohydrate with a small dose of vaccine. A single dose of TRIAD injected intraperitoneally induced a robust antibody response against carbohydrate without adverse effects. The generated response was mostly IgG and reached a maximum titer with one dose of the vaccine. We believe that this will allow us to generate a universal vaccine capable of protecting against all serotypes of N. meningitidis. This vaccine platform also readily lends itself to further modifications in that it is possible to include additional neisserial proteins.

Methods

All chemicals and solvents were purchased from commercial suppliers unless otherwise noted. CTAT was purified by recrystallization in ethanol/acetone prior to use.

Synthesis of N-Dodecanoylsuccinimide.

A solution of dodecanoic acid (0.539 mmol, 0.108 g), N-hydroxysuccinimide (NHS) (0.573 mmol, 0.0660 g), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HCl) (0.574 mmol, 0.110 g) in DMF was stirred at room temperature for 4 h (FIG. 18). The solution was diluted with ethyl acetate (20 mL) and washed with H20 (20 mL), saturated aqueous NaHCO3 (20 mL), and H20 (20 mL×2). The organic layer was dried over MgS04, filtered, and concentrated in vacuo. Purification after recrystallization (diethyl ether/hexane) afforded 0.0977 g (58%) of N-dodecanoylsuccinimide as white shiny crystals: Rr=0.85 (diethyl ether/hexane); mp 78-79 oc (lit. 15 78-81° C.); IR (thin film, NaCl) 2929 (m), 2852 (m), 1744 (s); 1H NMR (CDCh, 400 MHz) δ2.84 (s, 4H), δ 2.61 (t, J=8 Hz, 2H), δ 1.75 (m, J=8 Hz, 2H), δ 1.41 (m, 16H), δ 0.89 (t, J=8 Hz, 3H).

Synthesis of Dodecanoic Acid Tethered PADRE Peptide Conjugate.

To a solution of PADRE peptide (FIG. 17) (GenScript) (1.445 μmol, 1.950 mg, 2.890 μM) in 0.5 mL of DMSO-d6 was added a solution of N-dodecanoylsuccinimide (1.445 μmol, 0.430 mg, 2.89 μM) in 0.5 mL of DMSO-d6 followed by a solution of diisopropylethylamine (50 μL, 1.44 μmol, 28.7 mM) in DMSO-d6. The resulting solution was mixed well by vortexing and stirred at room temperature for 24 h. 1H NMR analysis of the reaction mixture indicated that approximately 85% of NHS C12-ester was reacted with PADRE by comparing the integration of starting material to product. The reaction mixture was concentrated in vacuo and stored at −20° C.

Isolation and Characterization of LOS.

The LOS was purified from N. gonorrhoeae F62˜ lgtD, a strain genetically modified to produce only the lacto-N-neotetraose LOS (L 7 immunotype), using a hot phenol/water extraction.

Synthesis and Characterization of Surfactant Vesicles.

Vesicles prepared with a molar excess of SDBS will be referred to as SDBS-rich (anionic). To prepare vesicles with the liposaccharides shown in FIG. 12 and FIG. 17, the liposaccharide was weighed directly into a vial containing 70.0 mg of SDBS (0.200 mmol) and 30.0 mg of CTAT (0.0658 mmol). Specifically, 1 mg of LOS or LPS and 0.2 mg of C12-PADRE conjugate were used to give a 10:1 w/w ratio of antigens in vesicles. Then 9.90 mL of water was added and samples were stirred for 60 min. Vesicles were purified from free conjugate by SEC, described previously. The incorporation of carbohydrate and C12-PADRE-conjugate were determined by colorimetric assays to determine the quantity retained in vesicle-containing fractions. The phenol/sulfuric acid colorimetric assay is described previously. The absorbance was measured at the Amax (˜490 nm) and compared to a standard curve, prepared for LOS and LPS to determine the total carbohydrate concentration in each sample.

Animal Trials.

Mice were immunized by the Stein lab intraperitonealy with either 10 μg of purified LOS alone, or 8.5 μg of conjugated vaccine (Vs-OS-PADRE). On day 21 and 42, mice were boosted with an equivalent amount of vaccine or oligosaccharide and blood samples were taken. Serum was recovered from all mice on day 51 in a terminal bleed.

Example 3 Catanionic Surfactant Vesicle Vaccine Fully Protects Mice Against Francisella tularensis LVS Challenge and Partially Protects Against Francisella tularensis Schu 54 Challenge

Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with “bare” vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS.

Methods

Vesicle Preparation and Purification.

The vesicle preparation method was adapted from a protocol developed by Kaler et al. and refined by Thomas et al. To prepare cultures for vaccine preparation, the stocks were grown to mid-log phase in Mueller-Hinton broth (MHB) (Becton, Dickinson Microbiology Systems), supplemented with 1% IsoVitaleX (Becton, Dickinson), 0.1% glucose (Sigma), and 0.025% ferric pyrophosphate (Sigma) at 37° C., while shaking. F. tularensis Schu S4 (FSC237; BEI Resources) was grown in Trypticase soy broth (TSB) (Becton, Dickinson Microbiology Systems) supplemented with 0.1% L-cysteine hydrochloride (Sigma), 0.1% glucose (Sigma), and 0.1% ferric pyrophosphate (Sigma) at 37° C., while shaking. To prepare 10 ml of vesicles, 25 ml (or 3 to 100 ml for experiments measuring the effects of protein loading on vesicle size and stability) of overnight F. tularensis cultures (optical density at 600 nm [OD600], 0.4 to 0.6) was pelleted by centrifugation and lysed in a 7.07-g/liter solution of the anionic surfactant sodium dodecylbenzenesulfonate (SDBS) (Tokyo Chemical Company, Ltd., Tokyo, Japan) in ultrapure endotoxin-free water (Sigma, St. Louis, Mo.). After 1 h, 30 mg of the ethanol-acetone recrystallized form of the cationic surfactant cetyltrimethylammonium tosylate (CTAT) (Sigma) per 10 ml of lysed bacteria was added, for a 3:1 molar ratio of SDBS to CTAT, and the vesicles were allowed to self-assemble overnight at room temperature with stirring. At this point, Schu S4-V preparations were plated on Mueller-Hinton blood plates and kept at 37° C. to ensure the sterility of the vaccine preparations for safe removal from the biosafety level 3 (BSL-3) laboratory. No colonies were detected on any of the plates (data not shown). The vesicle preparations were stored at 4° C. during this time. To separate the vesicles from free surfactants and cellular debris, the vesicle preparations were centrifuged to pellet large detritus, and the resulting supernatant was purified by size exclusion chromatography over a column composed of 10 ml of washed and packed Sephadex G-100 (Sigma). The vesicles remained stable at 4 to 25° C. for ≥5 months (data not shown). The protein content in the vesicles was determined by a modified version of the detergent-compatible (DC) protein assay with Reagent S (Bio-Rad, Hercules, Calif.), using bovine serum albumin (BSA) as a standard (Pierce, Rockford, Ill.). The vesicles varied in size, with radii ranging from 70 to 105 nm in bare vesicles and 80 to 120 nm in vesicles containing F. tularensis components, as determined by dynamic light scattering (DLS) with an LS-DYNA (Livermore Software Technology Corporation) set to a 90° angle, utilizing PhotoCore 5.3.8 analysis software for cumulants algorithm. Zeta potential was measured with a Malvern Zetasizer Nano ZS90, using phosphate-buffered saline (PBS) as an electrolyte source. Zeta potential measures surface charge on the vesicles and has been found to correlate with the stability of vesicles.

Immunization Protocol and F. tularensis Challenge.

Wild-type 6- to 8-week-old female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me.) and housed in a specific-pathogen-free facility at the University of Maryland Baltimore, Md., and in the animal BSL-3 (ABSL-3) suite at the University of Virginia. The mice were immunized with either the F. tularensis LVS (LVS-V) or the F. tularensis Schu S4 (S4-V) vesicles (35 μg protein) by either intraperitoneal (i.p.), intranasal (i.n.), or subcutaneous (s.c.) routes, as indicated, up to three times in 2-week intervals, with the last dose administered 14 days prior to live bacterial challenge. Where indicated, the resting period between the immunization and challenge was extended from 14 to 28 days. As a negative control, the mice received bare vesicles made in endotoxin-free water in the absence of bacterial pellets but purified identically to LVS-V and S4-V. For passive immunization experiments, the mice received 60 μl pooled serum samples from naive mice or from vesicle- or LVS-V-immunized animals by the intravenous (i.v.) route 1 day prior to challenge. The mice were challenged by the i.p. route with up to 100,000 CFU F. tularensis LVS or by the i.n. route with up to 50 CFU F. tularensis Schu S4. The challenge doses of F. tularensis LVS used in these studies were determined prior to each experiment to kill ≥80% of unimmunized mice.

The mice were checked twice daily for clinical symptoms of F. tularensis infection for 21 days following challenge. Clinical scores were assigned as follows: for mice infected with F. tularensis LVS, a score of 0 was assigned for healthy mice with normal behavior (exploring cage, feeding, alert), 1 for mild illness (usually marked by lower activity and weight loss), 2 for mild-moderate illness (mice showing symptoms of score 1 plus piloerection), 3 for moderate-severe illness (mice showing symptoms of score 2 plus assuming a hunched posture), 4 for severe illness (mice showing symptoms of score 3 and minimal activity or blepharitis [crustiness around the eye] involving one or both eyes), 4+ for moribund illness (mice showing symptoms of score 3 and were nonresponsive to stimulation [these mice were euthanized]), and 5 for mice found dead in their cage. Mice with scores of 4+ and 5 were reported on the day of death only and were excluded from analysis on subsequent days. The arithmetic means±the standard errors of the means (SEM) of the clinical scores were reported. For mice infected with the F. tularensis Schu S4 strain, a score of 0 was assigned for healthy mice with normal mouse behavior (exploring cage, feeding, alert), 1 for mild illness (decreased movement), 2 for moderate illness (decreased motion, eye closure), 3 for severe illness (motionless, eye closure, increased respirations, ruffled fur), and 4 for mice that were moribund (no motion in response to external stimuli plus meeting the criteria in reference 3) or dead. The moribund mice were euthanized.

To measure the blood serum antibody titers in F. tularensis LVS-infected mice, ˜200 μl blood was collected at the indicated intervals. Each mouse was euthanized and bled at the termination of the experiments. All animal experiments were conducted with Institutional Animal Care and Use Committee approval.

ELISA, Silver Stain, and Western Blots.

Enzyme-linked immunosorbent assays (ELISAs) were performed as previously described. Briefly, ELISA plates were coated with 2 μg/ml purified lipopolysaccharide from F. tularensis LVS (F. tularensis LVS LPS) or with 5±107 CFU/ml F. tularensis LVS. After washing, the plates were blocked with 10% fetal bovine serum (FBS). The serum samples were serially diluted in 10% FBS and 0.05% Tween 20 and were added to the coated plates for 90 min at 37° C. After extensive washing, horseradish peroxidase (HRP)-conjugated secondary antibodies (SouthernBiotech) were added to the wells at a 1:20,000 dilution. Detection was achieved using the 2,2=-azinobis(3 ethylbenzthiazolinesulfonic acid) (ABTS) system (Sigma) and the plates were read on a universal microplate reader, ELx800 (Bio-Tek Instruments, Inc.). The data were analyzed in MS Excel; the antibody titers were defined as the serum dilution corresponding to an average OD value of 3 standard deviations above the background OD values. The data for each mouse were graphed on a logarithmic scale, and the geometric mean was calculated for each group of mice at each time point. The geometric means were graphed separately in FIG. 22D for easier comparison.

Silver staining of membranes was performed as previously described. Briefly, vesicles and bacterial pellets were boiled in Laemmli sample buffer (with β-mercaptoethanol but without protease inhibitor cocktail) and separated by PAGE on Tris-glycine gels (12%, 10 to 20% gradient, or 4 to 20% gradient, as indicated) (Bio-Rad). The gels were fixed overnight in 40% ethanol, 0.5% glacial acetic acid, and washed for 5 min in 0.83% periodic acid (Sigma). After washing with water, the gels were stained with a basic solution of 0.8% silver nitrate (Sigma) for 15 min, washed extensively, and developed with 0.007% formaldehyde and 50 μg/ml citric acid for 10 min. The gels were washed with water, at which time they continued to develop and were scanned after 10 min.

For Western blots, vesicle and bacterial samples were boiled in Laemmli sample buffer and separated by PAGE as described above. The gels were transferred to an Immobilon-P membrane (Millipore) by wet transfer. The membranes were blocked in5% milk in Tris-buffered saline-Tween 20 (TBST) (Bio-Rad). Pooled serum samples from LVS-V- or Schu S4-V immunized mice were diluted 1:106 or 1:105, respectively, in 5% milk-TBST and incubated with the membranes overnight at 4° C. or 2 h at room temperature (RT). The dilutions of anti-LVS-V or anti-Schu S4-V were based on preliminary experiments that optimized the detection of bands in the F. tularensis lysates. After washing, HRP-conjugated human adsorbed goat-anti-mouse IgG1, IgG2b, IgG2c, and IgG3 secondary antibodies (Southern Biotech) were diluted 1:20,000 in 5% milk-TBST and incubated with the membranes for 60 min at RT. After extensive washing, the ECL Plus detection kit (GE Healthcare) was used to visualize the bands, which were recorded on BioMax MR film (Kodak). F. tularensis LPS-specific rabbit antibodies were kindly provided by Karen Elkins (FDA). Purified F. tularensis LVS DnaK and Tul4 were prepared as described previously.

Comparison of Different F. tularensis Strains by Western Blot Analysis.

F. tularensis strains Schu S4, MA00-2987, WY96-3418, KY99-3387, OR96-0246 (BEI Resources, Inc.) and F. tularensis LVS were grown in enriched TSB medium as described above. To estimate equal loading, each bacterial culture was grown overnight and was diluted to an OD600 of 0.300 prior to pelleting the bacteria and lysis in Laemmli sample buffer. PAGE, silver staining, and Western blot analysis were carried out as detailed above.

Real-Time PCR.

To assess the initial inflammatory responses, each mouse was euthanized 4 h after the first i.p. immunization with saline, vesicles, or LVS-V. Total RNA was extracted from the liver samples, and real-time PCR analysis was performed as previously described. In this study, we report the relative gene expression normalized to the expression of mouse hypoxanthine phosphoribosyltransferase (HPRT). All primer pairs used in this study have been published. The cytokine protein levels were measured by the Cytokine Core Laboratory (University of Maryland Baltimore [UMB]). Statistics. The data were analyzed with GraphPad Prism 4 for Windows (GraphPad Software, Inc., San Diego, Calif.). Specific tests include Fisher's exact test for comparisons of survival at day 14 post challenge, the log rank test for analysis of survival curves, the one-way analysis of variance (ANOVA), followed by a Tukey's post hoc test for analyses of multiple groups of data, and the Mann-Whitney test (nonparametric t test) for analyses of two groups of data.

Fuctionalization of Vesicles Used for Immunization in FIG. 20.

The T cell epitope PADRE was conjugated to dodecanoic acid employing the N-hydroxysuccinimide methodology described below. The lipid A portion of LPS is sufficiently hydrophobic to be incorporated into the vesicle leaflet, and therefore, no modification of LPS was required. Whole LPS isolated from Ft LVS (1) and/or the C12-modified PADRE peptide were incorporated during the assembly of catanionic vesicles as both surfactants were mixed concurrently at the same ratios as described in the text. These vesicles were administered to mice by i.p. injections twice, two weeks apart. Two weeks following the second immunization, mice were challenged with Ft LVS (FIG. 20) and survival of each individual mouse was followed for two weeks. Mice were bled prior to each injection and 11 days post-challenge. Antibody detection in sera were carried out by a FACS-based bead assay as previously described.

Conjugation of PADRE to a C12 Hydrophobic Tail.

A hydrophobic tail for the PADRE peptide was attached to the amino terminus as follows: A solution of dodecanoic acid (0.539 mmol, 0.108 g), N-hydroxysuccinimide (0.573 mmol, 0.0660 g), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HCl) (0.574 mmol, 0.110 g) in dimethylformamide (DMF) was stirred at room temperature for 4 h. The solution was diluted with ethyl acetate (20 mL) and washed with H2O (20 mL), saturated aqueous NaHCO3 (20 mL), and H2O (20 mL×2). The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. Purification after recrystallization (diethyl ether/hexane) resulted in 0.0977 g (58%) of N-dodecanoylsuccinimide ester as white, shiny crystals: Rf=0.85 (diethyl ether/hexane); mp 78-79° C. (lit.(3) 78-81° C.); IR (thin film, NaCl) 2929 (m), 2852 (m), 1744 (s); 1H NMR (CDCl3, 400 MHz) δ 2.84 (s, 4H), δ 2.61 (t, J=8 Hz, 2H), δ 1.75 (m, J=8 Hz, 2H), δ 1.41 (m, 16H), δ 0.89 (t, J=8 Hz, 3H). To a solution of PADRE peptide (GenScript) (1.445 μmol, 1.950 mg, 2.890 mM) in 0.5 mL of DMSO-d6 (deuterated) was added a solution of Ndodecanoylsuccinimide (1.445 μmol, 0.430 mg, 2.89 mM) in 0.5 mL of DMSO-d6 followed by a solution of diisopropylethylamine (50 μL, 1.44 μmol, 28.7 mM) in DMSO-d6. The resulting solution was mixed well by vortexing and stirred at room temperature for 24 h. 1H NMR analysis of the reaction mixture indicated that approximately 85% of the N-hydroxysuccinimide C12-ester was reacted with PADRE by comparing the integration of starting material to product. The reaction mixture was concentrated in vacuo and stored at −20° C.

Results

Immunization with F. tularensis LVS LPS-V Protects Against F. tularensis LVS but not F. tularensis Schu S4 Challenge.

We previously demonstrated that immunization with LPS from F. tularensis LVS (F. tularensis LVS LPS) only 2 days prior to live bacterial challenge fully protected mice against a lethal challenge with F. tularensis LVS. This protection was attributable to B1a cell- and antibody-mediated responses. The same regimen did not protect against F. tularensis Schu S4 challenge, despite the fact that both F. tularensis LVS and F. tularensis Schu S4 share the same O antigen, and therefore, antibodies should be cross-protective. In an attempt to improve the effectiveness of F. tularensis LVS LPS as a vaccine, we initially incorporated the purified F. tularensis LVS LPS, with or without a promiscuous The cell epitope called PADRE, into catanionic vesicles that were used as a delivery system. PADRE was covalently modified at the N terminus with a C12 hydrophobic tail via amide linkage (C12-PADRE) to enable its coupling to the vesicles. Catanionic vesicles form when two surfactants with oppositely charged head groups and sufficiently long hydrophobic tails (≥C10) are combined in the appropriate ratio (i.e., a molar ratio of 3:1 is ideal for SDBS and CTAT). Indeed, surfactant-based catanionic vesicles have been shown to be more stable than phospholipid-based vesicles. Mice immunized with two doses of purified F. tularensis LVS LPS (as a positive control) or with catanionic vesicles functionalized with F. tularensis LVS LPS (≥C12-PADRE), administered 2 weeks apart (FIG. 20A), survived i.p. challenge with F. tularensis LVS 2 weeks after final immunization (FIG. 20B). However, no protection was seen when similarly immunized mice were i.n.challenged with F. tularensis Schu S4. A serum IgM anti-LPS antibody response was induced in immunized mice, which significantly increased following challenge, regardless of the immunogen used (FIG. 20C). The purified F. tularensis LVS LPS also induced a detectable serum IgG anti-LPS response (FIG. 20C). The levels of IgG1 antibodies were significantly reduced in the vesicle-immunized mice, and to a lesser extent, IgG3 levels were also reduced compared to the controls (FIG. 20C). Therefore, we sought a new approach that would lead to a subunit vaccine that would both increase the IgG response and protect against infection with F. tularensis Schu S4.

Immunization with F. tularensis LVS-Vesicles Elicits Full Protection Against F. tularensis LVS Challenge.

Since F. tularensis LVS LPS-V failed to protect against F. tularensis Schu S4 challenge, we next modified our catanionic vesicle system in an effort to develop an improved F. tularensis subunit vaccine. We hypothesized that immunization with a subunit vaccine that contained a diverse assortment of the F. tularensis ligands, including hydrophobic and amphiphilic bacterial molecules, as well as bacterial DNA, would enable concurrent engagement of multiple innate and adaptive signaling pathways. We theorized that a strong pathogen-specific inflammatory response, coupled with a specific high-titer IgG anti-F. tularensis antibody response, would protect not only against F. tularensis LVS challenge but also against F. tularensis Schu S4 challenge. To this end, we prepared control vesicles by combining anionic and cationic surfactants in endotoxin-free water. To prepare vesicles containing F. tularensis components, the anionic surfactant was first added to pelleted cultures of F. tularensis LVS or F. tularensis Schu S4 to lyse the bacteria, and then the cationic surfactant was added to produce vesicles that incorporated bacterial components from the bacterial lysates. The resulting F. tularensis nanoparticles are referred to as LVS-V and Schu S4-V, respectively. No living organisms grew on Mueller-Hinton blood plates after contact with anionic surfactant (data not shown). The vesicles were subsequently purified over a Sephadex G-100 column to separate free bacterial components and unincorporated surfactants from the vesicles prior to immunizing the mice.

Initially, the mice were immunized twice by the i.p. route, 2 weeks apart, with sterile saline (as a control), LVS-V (35 μg protein), or an equivalent volume of bare vesicles as an additional control. Two weeks after the second immunization, all mice were challenged by the i.p. route with the indicated dose of F. tularensis LVS. Weight loss, clinical symptoms, and the survival of each mouse were monitored for 3 weeks. FIG. 21 shows the results of two separate experiments at this immunization schedule, with challenge doses of 30,000 CFU F. tularensis LVS/mouse (FIG. 1A to C) or 70,000 CFU F. tularensis LVS/mouse (FIG. 21 D to F). One experiment in which the resting period after immunization was extended to 4 weeks prior to challenge with 30,000 CFU F. tularensis LVS/mouse is also shown (FIG. 21 G to I). In the saline-immunized groups, mice suffered rapid weight loss (FIGS. 21 A, D, and G) and exhibited infection-associated clinical symptoms (FIGS. 21 B, E, and H) starting on the second day after infection and either expired or started to recover 5 to 6 days after infection. Seventy-five to 80% of the mice immunized twice with saline and challenged with F. tularensis LVS died when challenged with 30,000 (FIGS. 21 C and I) or 70,000 CFU F. tularensis LVS (FIG. 21 F). Immunization with LVS-V fully protected mice, as evidenced by the 100% survival rate at both challenge doses and the complete absence of weight loss and other clinical signs of disease, whether the resting time allowed between the final immunization and challenge was 2 weeks or 4 weeks (FIG. 21). Surprisingly, in both challenges carried out 2 weeks postimmunization, ≥80% of mice that were immunized with plain vesicles suffered only moderate weight loss during the peak infection period and survived F. tularensis LVS challenge (FIG. 1). Empty vesicle-immunized animals that were challenged 4 weeks after the second immunization exhibited more severe weight loss, similar to the saline controls (FIG. 21 G), suggesting that the effect of bare vesicle immunization might wane by 4 weeks postimmunization, but in contrast to the saline controls, the majority of these animals recovered (FIG. 21 I).

LVS-Vinduced Robust Antibody Responses, Including Isotype Class Switching.

To test whether immunization with LVS-V resulted in augmented humoral antibody responses, mice were immunized twice with saline, empty control vesicles, or LVS-V and challenged with 30,000 CFU F. tularensis LVS (challenge 1) by i.p. injection as described above, and then survivors, as well as a new group of saline-treated mice, were challenged i.p. with 50,000 CFU F. tularensis LVS (challenge 2) to determine if re-exposure would boost antibody responses in mice previously exposed to F. tularensis antigens by immunization and/or by the first challenge. Blood serum samples were collected prior to each immunization and challenge, as well as 1 week following the second challenge (FIG. 22A), and the antibody titers were measured by ELISA. To assess the levels of serum antibody directed against LPS and non-LPS epitopes, ELISA plates were coated with either purified F. tularensis LVS LPS or whole F. tularensis LVS (FIGS. 22 B and C). Isotype-specific antibodies were detected with secondary antibodies against mouse IgM (FIG. 22B) or a cocktail of secondary antibodies against all four mouse IgG subclasses (FIG. 22C).

While no F. tularensis-specific IgM response was observed in mice immunized with saline or vesicles (weeks 2 and 4), a low but significant IgM titer followed live bacterial challenge (weeks 7 and 8). Only mice immunized with LVS-V produced detectable levels of F. tularensis-specific antibodies prior to live bacterial challenge: IgM titers were detected as early as 2 weeks after the first immunization and increased after challenge with live F. tularensis LVS (weeks 7 and 8). The IgM antibody titers were similar when assayed on plates coated with either purified F. tularensis LVS LPS or whole F. tularensis LVS (FIG. 22B), indicating that essentially all of the IgM produced was directed against the LPS. Immunization with LVS-V, but not saline or bare vesicles, also induced antibody isotype switching to IgG. IgG titers were detectable 14 days after the first immunization, and were increased 2 weeks after the second immunization (FIG. 22C) when assayed by ELISA on plates coated with whole F. tularensis LVS. Notably, the prechallenge (week 4) antibody titers directed against whole bacteria exceeded anti-LPS IgG titers by >10-fold, indicating that the IgG responses were predominantly directed against non-LPS epitopes (FIG. 22D). Non-LPS-specific IgG antibodies remained strong and further increased to a titer of ˜106 following live bacterial challenge. Consistent with the responses to natural infections (12, 39), the antibody responses produced by immunization with saline or vesicles were detected only after live infection (week 7 and later) and were directed predominantly against the F. tularensis LPS. Thus, immunization of mice with LVS-V induces a strong IgG response to F. tularensis.

Passive Immunization Using Serum Samples from LVS-V-Immunized Mice Protects Against F. tularensis LVS Challenge.

Since we observed high antibody titers following LVS-V immunization directed predominantly against non-LPS epitopes, we sought to test whether these antibodies would be protective in F. tularensis LVS challenge. Donor mice were immunized as described above with either saline, LVS-V, or vesicles. Two weeks following the second immunization, serum samples were harvested and pooled according to the immunogen the donor mice received. It is noteworthy that these mice were not challenged with F. tularensis LVS. F. tularensis-specific IgM and IgG titers were measured by ELISA. Similar to the data in FIG. 2, serum samples from LVS-V-immunized mice contained significantly elevated levels of F. tularensis-specific IgG when assayed by ELISA against whole organisms (FIG. 23A). Sixty microliters of each serum pool was administered to naive mice by i.v. injection. The recipient mice were challenged 24 h later with 100,000 CFU F. tularensis LVS per mouse by the i.p. route, and weight loss, clinical symptoms, and survival were recorded for each mouse over the course of 2 weeks. Mice that received serum from saline-immunized mice (control serum) expired in 4 to 6 days (FIG. 23D). Mice that received serum from LVS-V-immunized mice experienced essentially no weight loss and much less severe clinical symptoms than controls (FIGS. 23 B and C). Six of seven of these animals (86%) recovered in the second week following infection (FIG. 3C) (P=0.0002 compared to control serum recipients). Mice that received serum from bare vesicle-immunized mice showed a slight delay in the onset of clinical symptoms (FIG. 23C) but mirrored the control serum recipients with respect to weight loss (FIG. 23B). Five of seven recipients of sera from empty vesicle-immunized donors died with the same kinetics as the control serum recipients (FIG. 23D). These data suggest that the survival in F. tularensis LVS challenge correlates with IgG anti-F. tularensis antibody titer. Despite protection against F. tularensis LVS challenge, serum from an LVS-V-immunized donor, transferred i.v., did not protect against an i.n. challenge with 50 CFU F. tularensis Schu S4 (data not shown).

LVS-V Induces Cytokine Gene and Protein Expression In Vivo.

Because partial protection of mice that were immunized with empty vesicles was observed in response to lethal F. tularensis LVS challenge (FIG. 21), despite antibody responses that were no different than those of naive animals (FIG. 22), we sought to test the inflammatory potentials of the surfactant vesicles, as this is often a property of adjuvants. Mice were injected i.p. with either saline, bare vesicles, or LVS-V at the same dose as used for immunization. Four hours later, each mouse was bled and euthanized and the livers harvested for reverse transcription-quantitative PCR (qRT-PCR) analysis of proinflammatory gene expression. Compared to the saline controls, LVS-V induced significantly higher levels of the following genes: keratinocyte chemoattractant (KC), 58-fold (P=0.0061); monocyte chemoattractant protein 1 (MCP-1), 5.7-fold (P=0.0061); tumor necrosis factor alpha (TNF-α), 5.3-fold (P±0.0061); and IL-1β, 4.6-fold (P_0.0061) (FIG. 4A). IFN-β, IFN-γ, gamma interferon inducible protein 10 (IP-10), RANTES (data not shown), IL-6, and inducible nitric oxide synthase (iNOS) (FIG. 24A) were induced in only some of the LVS-V-treated mice by 4 h, and consequently, the mean induction values for these genes were not statistically significantly different compared to those of the saline controls. Only a few of these inflammatory genes were induced in the livers of mice treated with plain vesicles and to a much lower level than those induced by LVS-V (FIG. 24A). KC and IL-6 proteins were also detected in the serum samples of mice 4 h after immunization with LVS-V but decreased rapidly thereafter (FIG. 24B). No difference was observed in the Th2-inducing cytokines IL-4 and IL-13 (data not shown). Spleens were also harvested 4 h after injection and showed similar expression patterns but lower levels of induction of proinflammatory genes (data not shown).

Reproducibility of Size and Stability of F. tularensis Nanoparticles.

To optimize our immunization protocol, we tested whether the size or stability of the F. tularensis nanoparticles was affected by the protein content of the vesicles. F. tularensis LVS pellets containing increasing numbers of bacteria (from ⅛-fold below up to 4-fold above the standard input concentration of 25 ml of overnight culture) were processed into vesicles using the identical amounts of surfactants and water for each preparation, as described in Methods. Purified nanoparticles were analyzed for protein content and physical characteristics. Measurement of the zeta potential, a measure of vesicle stability, revealed that vesicles that incorporated more bacterial components (as evidenced by increased protein concentration of the purified vesicle preparations) exhibited lower surface charges (FIG. 25A). Lower surface charge is typically associated with the destabilization of vesicles, yet no differences in toxicity were observed between mice immunized with vesicles of low versus high surface charge (data not shown). The average vesicle size (˜80-nm radius) was also not significantly affected by the amount of bacterial starting material incorporated in the vesicles (FIG. 25B). Importantly, different batches of F. tularensis nanoparticles prepared from either F. tularensis LVS or F. tularensis Schu S4 using the standard protocol of 25 ml of overnight bacterial culture exhibited a high consistency of physical parameters, such as average radius and surface charge (FIGS. 25 A and B).

F. tularensis LVS LPS and Proteins, Including Epitopes Recognized by Immune Mouse Serum, are Extracted into Vesicles with a High Degree of Consistency.

FIG. 26A shows a silver stained gel in which two independently prepared batches of empty vesicles and LVS-V (designated batch 1 and batch 2), as well as lysed samples of the F. tularensis LVS pellets from which the respective LVS-V batches were produced, were electrophoresed side by side. Silver staining revealed that there were no detectable bands in the bare vesicles, whereas most F. tularensis LVS proteins were consistently extracted into LVS-V preparations (FIG. 26A). Some bands, such as the one with an apparent molecular mass of ˜20 kDa, were enriched in the vesicles, while other proteins were less efficiently incorporated. Western analysis using a polyclonal anti-F. tularensis LPS antibody shows that F. tularensis LPS is also incorporated into LVS-V, as evidenced by the typical LPS ladder FIG. 27). Bands detected by Western analysis using serum samples from immunized mice before and after F. tularensis LVS challenge are also consistently incorporated into LVS-V (FIG. 26B). The vesicles that were made in the absence of F. tularensis LVS and served as control immunogens were also free from contamination, as evidenced by the absence of bands in the silver stain and Western analyses (FIG. 26). Similar batch-to-batch uniformity was observed in vesicles extracted from F. tularensis Schu S4 by silver stain and Western analysis (FIG. 27).

Antibody Epitope Specificity Matures Following Infection with Live F. tularensis LVS.

FIG. 26C also illustrates maturation of the IgG antibody response in mice that were immunized with F. tularensis nanoparticles, as evidenced by a comparison of the diversity of antigens detected by Western analysis in serum samples collected before and after challenge with F. tularensis LVS. Using serum samples collected immediately prior to challenge (two weeks after the second immunization with LVS-V) and a secondary anti-mouse IgG antibody, 5 prominent bands were detected in the LVS-V samples, one of which exhibited an apparent molecular mass of ˜55 kDa and was the predominant species detected by Western analysis. Minor bands with molecular masses of ˜50 kDa, ˜40 kDa, ˜20 kDa, and ˜12 kDa were also consistently detected at the same exposure. Immunoreactive bands of ˜95 kDa, ˜45 kDa, ˜37 kDa, ˜33 kDa, and ˜10 kDa were also present in LVS-V but were detected only after a long exposure (data not shown). A very low-molecular-weight species was detected exclusively in the F. tularensis LVS whole-cell lysates by Western analysis with both the anti-LPS antibody (FIG. 27) and the LVS-V immune serum (FIG. 26B).

Among the low-molecular-weight bands detected in silver staining, none were found that corresponded to the F. tularensis LVS lysate-specific band. In serum samples collected 1 week following a second F. tularensis LVS challenge, IgG responses to the minor band at ˜12 kDa were greatly enhanced to reveal a second immunodominant band. The responses to all other bands were also enhanced to various degrees, with the greatest increase seen at bands with apparent molecular masses of ˜95 kDa, ˜37 kDa, and ˜12 kDa. After the exposure to live bacteria, new minor bands with apparent molecular masses of ˜86 kDa, ˜67 kDa, and ˜22 kDa were detected. This same pattern was seen in both batches of LVS-V and F. tularensis LVS lysates. The same bands were detected regardless of whether LVS-V was produced in the absence or presence of protease inhibitors (FIGS. 27 B and C).

In the next series of experiments, blood serum samples were pooled from mice vaccinated with LVS-V (i.p./i.p.) and challenged with F. tularensis LVS (i.p.) and from mice vaccinated with Schu S4-V (i.p./i.n.) and challenged with F. tularensis Schu S4 (i.n.). In preliminary studies, we found that a 1:1,000,000 dilution of anti-LVS-V serum and a 1:100,000 dilution of the anti-Schu S4-V serum gave equivalent results in ELISA (data not shown). These dilutions were chosen for Western analysis, where they also detected several bands with the same intensity. A recent F. tularensis LVS subunit vaccine composed of Tul4 and DnaK successfully protected mice against intranasal challenge with F. tularensis LVS. Indeed, both purified Tul4 and DnaK preparations were detected with antisera from mice immunized and challenged homologously with either LVS-V and F. tularensis LVS or Schu S4-V and F. tularensis Schu S4 (FIGS. 28 A and B). The anti-Schu S4 serum reacted more strongly with Tul4 than the anti-LVS serum (note in FIG. 28B that these were exposed for only 1 s, because a longer exposure of the Tul4 protein detected by the anti-Schu S4-V antiserum was grossly overexposed). No bands were detected in bare vesicles with either antiserum preparation (FIGS. 28 A and B). Five predominant bands (including one that appears to be a doublet) were detected by the two antisera: the anti-LVS-V serum detected bands at molecular masses of ˜55 kDa, ˜40 kDa (doublet), ˜20 kDa, ˜12 kDa, and a faint band at ˜90 kDa (FIG. 28A). Interestingly, the anti-Schu S4-V antiserum detected the same bands but differed in the intensity of the bands. Specifically, the ˜12-kDa band was poorly detected, the ˜20-kDa species was more strongly detected, and the ˜90-kDa band, while still faint, was more apparent than in blots developed using the anti-LVS-V antiserum (FIGS. 28 A and B). Taken together, these data confirm our finding that the antibody response matures following exposure to live bacterial challenge (FIG. 26) and they extend it by showing that several immunodominant species are differentially detected by serum produced in response to LVS-V immunization/F. tularensis LVS challenge versus Schu S4-V immunization/F. tularensis Schu S4 challenge.

F. tularensis Epitopes Recognized by Either LVS or Schu S4 Antiserum are Found in Other F. tularensis Strains as Well.

We sought to test whether the epitopes recognized by the immune sera from LVS-V-immunized LVS-challenged mice and Schu S4-Vimmunized Schu S4-challenged mice would be present in other strains, including some that are clinically relevant. F. tularensis strains MA00-2987 (A1 strain), WY96-3418 (A2 strain), and KY99-3387 and OR96-0246 (B strains) were grown in enriched TSB. The samples were subjected to Western analysis with the antisera described above. Approximately equal loading was confirmed by silver staining (data not shown). Prebleed serum failed to detect any bands by Western analysis (data not shown). Serum samples collected 2 weeks after the second immunization with LVS-V (i.p./i.p.) but prior to F. tularensis LVS challenge (week 4) (FIG. 29A), after i.p. challenge with F. tularensis LVS (week 8) (FIG. 7B), or after immunization (i.p./i.n.) with Schu S4-V and i.n. challenge with Schu S4 (FIG. 29C) each showed remarkable consistency in their binding patterns across different F. tularensis strains. Interestingly, antiserum from the LVS-V-immunized F. tularensis LVS challenge survivors recognized an additional high-molecular-weight band in the type A1 (Schu S4, MA00) and A2 (WY96) strains (molecular mass, ˜105 kDa) not detected in the type B strains (F. tularensis KY99, OR96, and LVS).

LVS-V and Schu S4-V Immunization Partially Protect Against i.n. Schu S4 Challenge.

The most pressing need for a Francisella vaccine is for the protection of populations in case of aerosol releases of virulent type A strains of F. tularensis. Since LVS-V appears to be proinflammatory early after immunization and induces robust anti-F. tularensis antibody titers, we sought to test whether the F. tularensis nanoparticles might also protect against F. tularensis Schu S4, the most extensively studied model strain of the type A Glade. Initially, the mice were immunized as before (i.p./i.p., 2 weeks apart) with either sterile PBS, LVS-V, or the same volume of empty vesicles. Two weeks following the second immunization, all mice were challenged i.n. with ˜10 CFU F. tularensis Schu S4. The mice immunized with PBS rapidly developed tularemia, and all either died or had to be euthanized by the end of day 5 post challenge. In contrast to LVS challenge, mice that had been immunized with empty vesicles were not protected against Schu S4 challenge and developed clinical symptoms with similar kinetics as the saline-immunized mice and exhibited a nonsignificant delay in time to death. However, in mice vaccinated with LVS-V, we observed a delay in the onset of clinical symptoms, and one of five mice survived (data not shown).

To improve on the partial protection gained by LVS-V immunization against Schu S4 challenge, the mice were next immunized with Schu S4-V rather than LVS-V. Utilizing the same immunization regimen as for LVS-V with Schu S4-V (i.p./i.p., 2 weeks apart), only one of 10 mice survived i.n. challenge with ˜20 CFU F. tularensis Schu S4. However, the onset of symptoms and time to death from tularemia were delayed by almost 2 days, which was significant by the Mann-Whitney test (nonparametric t test) compared to animals that had been immunized with vesicles only (FIG. 30).

Heterologous Routes of Immunization with Schu S4-V Result in Improved Protection Against Schu S4 Challenge.

Since the number of immunizations and the route of administration affect the strength and type of the subsequent immune response, we compared outcomes from Schu S4 challenge following immunization with either 2 or 3 doses, as well as by diverse administration routes. The results from 5 independent experiments are summarized in Table 1. In each experiment, the control group of mice was immunized with bare vesicles, and all of these mice died within 5 days (on average), even with a challenge dose as low as 3 CFU F. tularensis Schu S4 per mouse. All mice that received Schu S4-V by i.p. and/or by i.n. route displayed a delayed onset of symptoms and delayed time to death by 1 day. In mice immunized via the subcutaneous (s.c.) route, the time to death was delayed less but was still statistically significant. No mice survived challenge after two immunizations with Schu S4-V by combinations involving the s.c. route or i.n. route only. Improved partial protection was gained through heterologous prime-boost using i.p./i.n. and i.n./i.p. routes. In the single most effective experiment, four of six mice (66%) that were immunized with Schu S4-V i.p./i.n. survived challenge with ˜22 CFU live F. tularensis Schu S4 (data not shown). The clinical scores of a representative experiment are shown in FIG. S3 in the supplemental material and illustrate the effects of the various immunization regimens. The addition of a third i n immunization also improved the outcome of challenge, assessed by a slight improvement in the fraction of survivors and further delay in time to death in nonsurvivors (FIG. 30; FIG. 31A). Systemic F. tularensis specific IgG titers were also increased following a third i.n. immunization (FIG. 31B). Heterologous immunization routes (i.e., i.n. and i.p. administration, with two immunizations) showed similar titers compared to three doses administered solely through the i.n. route, and the administration of a third i.n. dose further increased IgG titers. Regardless of the route of immunization, the blood serum IgG levels of mice that survived Schu S4 i.n. challenge (bled 21 days postchallenge) were approximately 10-fold higher than those of nonsurvivors (bled 5 to 8 days postchallenge at the time of euthanasia) (i.e., average titers of ˜125,000 and ˜12,000, respectively). Together, these data show that significant partial protection against virulent type A challenge can be achieved with two doses as a heterologous (i.p./i.n.) prime-boost immunization or by three i.n. immunizations with Schu S4-V.

Antigen Identification.

In this experiment, cell lysates from Francisella tularensis LVS mutant strains were subjected to SDS-PAGE followed by Western analysis using sera from mice immunized twice with either LVS-V or Schu S4-V by intraperitoneal injections. The sera from our immunized mice failed to detect discrete bands (circles) corresponding to FopA and Tul4 proteins (FIG. 47).

Example 4 Drug Delivery Application for Catanionic Surfactant Vesicles

There is interest in extending the role of catanionic vesicle systems for drug delivery application in order to provide an alternative to their liposomal counterparts. Catanionic vesicles can be easily prepared, are stable for yeast, and can be sterilized by autoclaving. Furthermore, catanionic vesicles can be functionalized, allowing the ability for targeted druf delivery of a payload to specific tissues. Toward this goal, we studied catanionic vesicles loaded with the drug candidates doxorubicin, maytansine, and paclitaxel. A for targeted delivery applications, we studied the cytotoxicity of doxorubicin-loaded catanionic vesicles fuctionalized with a targeting agent.

Methods

Doxorubicin.

To prepare doxorubicin-loaded vesicles, an aqueous solution of doxorubicin was prepared. To increase solubility of doxorubicin, the solution was sonicated in a water bath until full dissolution. Then, 70.0 mg of SDBS (0.200 mmol) and 30.0 mg of CTAT (0.0658 mmol) was weighed into the vial followed by the addition of 9. 90 mL of the doxorubicin solution and stirring for 60 min. Vesicles were purified from free drug by SEC and analyzed by UVNIS. A 0.5 mL portion of each vesicle fraction was transferred to an empty vial where 0.5 mL absolute ethanol was added to the sample to prevent light scattering during absorption measurements. The samples were vortexed and the absorbance was measured at 480 nm.

Maytansine.

To prepare maytansine vesicles, maytansine was dissolved in THF (5.1 mg/mL). A 196 μL (1.00 mg) aliquot of the solution was added to a vial containing 70.0 mg of SDBS (0.200 mmol) and 30.0 mg of CTAT (0.0658 mmol). Then, X mL of water was added and stirred for 60 min. Vesicles were purified from free drug by SEC and analyzed by UVNIS. A 0.5 mL portion of each vesicle fraction was transferred to an empty vial where 0.5 mL absolute ethanol was added to the sample to prevent light scattering during absorption measurements. The samples were vortexed and the absorbance was measured at 290 nm.

Paclitaxel.

To prepare paclitaxel vesicles, 0.5 mg of paclitaxel was dissolved in 9.90 mL (0.200 mmol) of an SDBS solution (7.07 mg/mL). The solution was stirred for 60 min. Then 30.0 mg of CTAT (0.0658 mmol) was added and stirred for 60 min. Vesicles were purified from free drug by SEC and analyzed by UVNIS. A 0.5 mL portion of each vesicle fraction was transferred to an empty vial where 0.5 mL absolute ethanol was added to the sample to prevent light scattering during absorption measurements. The samples were vortexed and the absorbance was measured at 445 nm.

Results

In order for catanionic vesicles to be used in drug delivery, their loading efficiency and toxicity were studied. In addition, drug loaded catanionic vesicles were functionalized with targeting agents toward cell lines that over express the receptor specific to the targeting agent. Cell toxicity was compared between targeted and untargeted catanionic vesicles in order to determine if targeting increased drug uptake into cells (FIG. 32).

Doxorubicin Loaded Catanionic Surfactant Vesicles.

Catanionic surfactant vesicles were loaded with the drug doxorubicin in order to determine the retention of the drug over time. Vesicles were loaded with 19 μg/mL doxorubicin (33 μM) and an aliquot of the vesicle stock suspension was purified by SEC each day over 15 days. Vesicles were 140±10 nm in diameter and their size did not change over the 15 day timeframe, as determined by DLS. These results indicated that catanionic vesicles loaded with doxorubicin are larger in size when compared to unloaded vesicles (˜120 nm) and remained stable over time.

Vesicle-containing fractions from each day were disrupted with ethanol and analyzed by UVNIS. Catanionic vesicle fractions purified on different days all showed complete retention of doxorubicin (FIG. 33). These results indicated that doxorubicin was loaded into catanionic vesicles and that these systems did not leak drug over time.

Since vesicles were proven to incorporate and retain doxorubicin at low concentrations, we wanted to determine the maximum loading of drug in vesicles. Catanionic vesicle stock solutions were prepared containing 100 μg/mL (172 μM), 150 μg/mL (259 μM), 200 μg/mL (345 μM), and 300 μg/mL (517 μM) of doxorubicin. When using high concentration stock solutions of doxorubicin (200 μg/mL and 300 μg/mL), vesicles formed but a red precipitate was observed. Catanionic vesicles containing these concentrations were centrifuged and the precipitate was removed. Suspensions were purified by SEC and vesicle containing fractions eventually formed a red precipitate. These result indicated that doxorubicin gradually leaked out of vesicles. If full retention of the drug is require, catanionic vesicles cannot be loaded with >200 μg/mL (345 μM) of doxorubicin.

Vesicles prepared with 100 μg/mL and 150 μg/mL doxorubicin stock solutions were stable over an extended period and did not precipitate drug, even after purification by SEC (FIG. 34). These results showed that vesicles could be initially loaded with a maximum of 150 μg/mL of doxorubicin and after SEC contained 88 μg/mL of doxorubicin.

After determining the maximum loading of doxorubicin into catanionic vesicles, we were interested in studying the toxicity of these drug formulations. As a benchmark for toxicity in humans, a typical dose of doxorubicin is 40-60 mg/m2. For a typical person, this amount equates to a total dose of from about 150 to about 200 mg of doxorubicin. The dose limiting toxicity (DLT) for doxorubicin is myelosuppression where nausea, vomiting, cardiotoxicity, and alopecia are other side effects. In order to determine toxicity of doxorubicin loaded catanionic vesicles compared to the free drug, catanionic vesicles were loaded with doxorubicin and incubated in several different cell lines. The WST-1 cell proliferation assay was used to measure the cytotoxic effects of catanionic vesicles.

The cytotoxicity of catanionic vesicles was initially studied in the human hepatocellular carcinoma cell line HepG2. These cells exhibit many of the characteristics of normal liver cells. Since many drugs are toxic to the liver, these cell lines are used for screening the cytotoxicity of new drugs. HepG2 cells were used to study doxorubicin loaded catanionic vesicles to determine if the toxicity of the drug in vesicles was less than free drug. The WST −1 cell proliferation assay was used to determine whether formazan dye was taken up by live cells. In this assay, mitochondrial enzymes of living cells break down the dye so that the product absorbs at a specific wavelength. In other words, when cells are proliferating, formazan dye accumulates and when cells are not proliferating, dye levels drop.

Results of toxicity studies in HepG2 cells yielded an IC50 of 51 μg/mL for DOX-loaded vesicles and an IC50 of 0.16 μg/mL for free DOX (FIGS. 35 and 36). These results indicated a 300-fold reduction in doxorubicin's IC50 values compared to free drug. Therefore, the toxicity of doxorubicin is greatly reduced when loaded into catanionic vesicles.

Next, we wanted to determine the toxicity of bare vesicles (unloaded vesicles) in HepG2 cells. The IC50 of bare catanionic vesicles was 58 μg/mL (FIG. 35). This IC50 value indicated that bare and DOX-loaded catanionic vesicles have the same cytotoxicity. Therefore, the toxicity of DOX-loaded vesicles resulted from the catanionic vesicles themselves and not from DOX. Therefore, DOX-loaded and bare vesicles showed that the toxicity of doxorubicin is completely removed when loaded within catanionic systems (FIG. 37).

Targeted Doxorubicin Loaded Catanionic Surfactant Vesicles.

Since DOX-loaded and unloaded catanionic vesicles have low toxicity in normal liver cells, we wanted to study these systems with the addition of a targeting moiety. We studied targeted DOX-loaded catanionic vesicles to determine if toxicity increased as a result of uptake into cells via targeting agent-receptor binding (FIG. 38).

DOX-loaded catanionic vesicles were functionalized with C12-folate conjugate (FIG. 39). Cytotoxicity of targeted DOX-loaded vesicles was determined in two different cell lines. Cells were grown in folate-depleted media so that the folate in media did not compete with the folate-targeting moiety on vesicles. A549 cells were chosen for initial studies because they minimally express the folate receptor. Cytotoxicity IC50 values of targeted bare vesicles and targeted-DOX vesicles in HepG2 cells were 7.2 μg/mL and 5.6 μg/mL, respectively (FIG. 40). Therefore, both targeted-unloaded and targeted DOX-loaded vesicles had similar toxicities.

Next, we wanted to determine the cytotoxicity of these systems when incubated in IGROV −1 cells. These cells were chosen because they highly express the folate receptor. Cytotoxicity IC50 values of targeted bare vesicles and targeted-DOX vesicles were 3.4 μg/mL and 2.2 μg/mL, respectively (FIG. 41). These results indicated that targeted vesicles were not more toxic when incubated with cells that over express the specific receptor.

Results from toxicity studies with catanionic vesicles did not show increased toxicity of targeted catanionic vesicles. However, there could be several explanations as to why toxicity did not increase: 1) catanionic vesicles were not loaded with enough targeting agent or 2) catanionic vesicles were not taken up by cells. In order to study the latter possibility, future work will be devoted to determine whether catanionic vesicles enter cells. Catanionic vesicles will be loaded with a dye and functionalized with a targeting agent. Fluorescently labeled catanionic vesicles will be incubated with cells and studied by microscopy to observe the rate at which catanionic vesicles with and without a targeting agent are endocytosed by cells (FIG. 42).

Maytansine Loaded Catanionic Suifactant Vesicles.

Maytansine is an extremely toxic drug that is insoluble in water and most solvents (FIG. 43). Catanionic vesicles were loaded with the neutral hydrophobic drug maytansine in the hope that the drug could be solubilized and incorporated for drug delivery. Maytansine was solubilized in THF and then added during vesicle formation. This solubilization was required in order for maytansine to dissolve so that the drug could be incorporated into catanionic vesicles. Vesicles had a hydrodynamic radius of 126±3 nm and were purified by SEC. All fractions were disrupted with ethanol and measured by UV NIS. Unfortunately, maytansine does not contain a good chromophore (FIG. 43). Maytansine has an absorbance at 290 nm, which is close to the absorbance of the surfactants used in our catanionic vesicles. UV NIS of the vesicle-containing fraction showed a peak at 290 nm and was compared to bare vesicle containing fractions (FIG. 44). It was difficult to officially determine whether the drug was successfully incorporated since the absorbance of maytansine and the surfactants absorb light in the same region. For this reason, a drug should be modified with a fluorescent tag so that incorporation into vesicles can be determined.

Paclitaxel Loaded Catanionic Surfactant Vesicles.

Since the lack of a chromophore made it difficult to characterize it loading in catanionic vesicles, vesicles were loaded with a drug containing a fluorescent label. Paclitaxel fluorescein derivative (FIG. 45) was added to vesicles prepared with dry surfactants and water. These vesicles formed but did not yield yellow suspensions. Due to the insolubility of paclitaxel, the drug was not incorporated during vesicle formation. Therefore, a solution of SDBS was used to solubilize paclitaxel by incorporating the drug into the hydrophobic region of micelles. Then, solid CTAT was added, and yellow catanionic vesicles formed. After purification by SEC, vesicle-containing fractions remained yellow in color. These results indicated the presence of paclitaxel within catanionic vesicles, likely by the initial incorporation of the drug into micelles followed by incorporated into the bilayer leaflet after the addition of the second surfactant. Fractions were disrupted with ethanol and their absorbance was measured at 445 nm. Results of UV NIS show that paclitaxel conjugate was successfully incorporated in vesicles (FIG. 46).

Catanionic vesicles can be loaded with drug molecules and functionalized with targeting agents. Catanionic vesicles loaded with doxorubicin showed low toxicity in the presence of normal liver cells. Catanionic vesicles could be used for drug delivery of doxorubicin similar to the liposomal formulation Doxil. Targeted catanionic vesicles did not increase toxicity of drug-loaded vesicles, but further studies will be performed in order to increase the targeting agent and to determine whether catanionic vesicles are incorporated into cells. We have also shown incorporation of other drugs into catanionic vesicles. Fluorescently labeled drugs allow for better characterization in catanionic vesicles.

Example 5 Methods for Whole Cell Extraction with Catanionic Vesicles

Here we describe how catanionic surfactant vesicles can extract membrane components from the Gram negative pathogen Neisseria gonorrhoeae using alternate methodologies. It is advantageous to have methods that have a preference for the incorporation of higher or lower molecular weight molecules.

Methods

In the case of Neisseria gonorrhoeae, further modifications were performed by modifying the fourth extraction methodology into two methods (see FIG. 1 for reference), methods 4-I and 4-II. These methods do not result in complete cell lysis. The cells were spun down in media and the supernatant was removed, leaving the pellet in the bottom.

In Method 4-I, 30 mg of CTAT, 71 mg SDBS, 6.9 mL of Millipore water, and 3 mL of cell suspension were mixed and then stirred for 15 minutes. The mixture was centrifuged for 1 hour and the supernatant was decanted. 1 mL of the sample of supernatant was purified on the column.

In Method 4-II, 9.9 mL of preformed vesicles (1% SDBS rich), 3 mL cell suspension, and 5.91 mL of Millipore water were mixed and then stirred for 1 hour. The mixture was centrifuged for 1 hour and the supernatant was decanted. 1 mL of the sample of supernatant was purified on the column.

Results

The amounts of protein and carbohydrate collected for each method are shown in FIG. 47. The proteins preferentially extracted using the various methods were further analyzed by gel electrophoresis. Silver staining of vesicles derived from extraction of Neisseria gonorrhoeae by Methods 4-I and 4-II are shown in FIG. 48.

The images of Neisseria gonorrhoeae gels show that the extraction methodologies are extracting different proteins. The main differences in the gel are marked in FIG. 48. Method 4-II seems to have extracted higher concentrations of low molecular weight proteins while Method 4-I seems to have extracted higher concentrations of higher molecular weight proteins. This seems to be consistent with the results from colorimetric and BCA assays because, as can be seen from FIG. 47, Method 4-II extracts more carbohydrates, rather than proteins, than does Method 4-I. Some of the bands that seem to show up in F62ΔlgtD strain from Neisseria gonorrhoeae Methods 4-I and 4-II seem to be from porin, multiple lipoproteins, and LPS, all known cell surface components. Method 4-II seems to be extracting more carbohydrate content than protein content from Neisseria gonorrhoeae. 

What is claimed is:
 1. A composition comprising a catanionic surfactant vesicle; wherein the catanionic surfactant vesicle comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens from whole bacterial cell extract; wherein the at least one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, or at least one membrane-bound polypeptide or at least one combination of a membrane-bound sugar and membrane-bound polypeptide; and wherein the membrane-bound sugar, the membrane-bound polypeptide, or the combination of the membrane-bound sugar and membrane-bound polypeptide is positioned at least partially in the unilamellar bilayer; and wherein the catanionic surfactant vesicle comprises bacterial lipooligosaccharide (LOS) or lipopolysaccharide (LPS) and one or a plurality of bacterial lipoproteins or bacterial membrane-bound protein; and wherein the catanionic surfactant vesicle comprises a percentage of carbohydrate from about 0% to about 25% by weight.
 2. The composition of claim 1, wherein the at least one or plurality of bacterial immunogens is from a Gram-negative bacterial cell.
 3. The composition of claim 1, wherein the at least one or plurality of bacterial immunogens is derived from cells that are from Neisseria or Francisella.
 4. The composition of claim 1, wherein the catanionic surfactant vesicle comprises a percentage of carbohydrate from about 0.1% to about 16% by weight.
 5. The composition of claim 1, wherein the one or plurality of bacterial immunogens comprise one or a combination of pilin, porA, porB, OPA, DnaK, Tul4, and FopA.
 6. The composition of claim 1, wherein the one or plurality of bacterial immunogens comprises Pan HLA-DR reactive epitope (PADRE) individually or conjugated to one or a plurality of membrane-bound sugar molecules.
 7. The composition of claim 1, wherein the catanionic surfactant vesicle is lyophilized.
 8. A pharmaceutical composition comprising: the composition of claim 1 and a pharmaceutically acceptable carrier.
 9. A vaccine comprising: (i) a plurality of catanionic vesicles, wherein at least one of the catanionic vesicles comprises a unilamellar bilayer comprising a mixture of oppositely charged single-tailed surfactants and one or a plurality of bacterial immunogens from whole bacterial cell extract wherein the one or plurality of bacterial immunogens comprise at least one membrane-bound sugar, or at least one membrane-bound polypeptide or at least one combination of a membrane-bound sugar and a polypeptide; wherein the membrane-bound sugar, the membrane-bound polypeptide or the combination of the membrane-bound sugar and the membrane-bound polypeptide is positioned at least partially in the unilamellar bilayer, and (ii) a pharmaceutically acceptable carrier; wherein the one or plurality of bacterial immunogens is from Neisseria or Francisella cells; and wherein the catanioinc vesicles comprise a diameter of 1000 nm or less.
 10. The vaccine of claim 9 further comprising one or a plurality of: (i) a nucleic acid molecule encoding one or a plurality of immunogens; (ii) a nucleic acid molecule encoding one or a plurality of immunomodulating proteins; (iii) a protein adjuvant; (iv) a synthetic adjuvant; (v) an attenuated bacterial cell; (vi) a bacterial cell comprising a nucleic acid molecule encoding one or a plurality of immunogens; or (vii) a bacterial cell comprising a nucleic acid molecule encoding one or a plurality of immunomodulating proteins.
 11. A method of treating and/or preventing establishment of a pathogen infection in a subject in need thereof, comprising: administering to the subject the pharmaceutical composition of claim
 8. 12. A method of immunizing a subject comprising: administering to a subject in need thereof an amount of the vaccine of claim 9 effective to elicit an immune response.
 13. The method of claim 12, wherein the immune response is a protective, antigen-specific immune response.
 14. A method of integrating a bacterial antigen into a vesicle comprising: (a) centrifuging bacterial cells comprising at least one bacterial antigen; (b) removing any bacterial growth medium; (c) adding a weight/weight molar ratio of cationic surfactant and anionic surfactant of either about 30:70 or about 70:30 in aqueous solution to the bacterial cells; (d) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification or removal of bacterial sugar molecules prior to steps (a)-(d).
 15. The method of claim 14, wherein the cationic surfactant and anionic surfactant are sodium dodecyl benzene sulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT), respectively, at a molar weight ratio of about 70:30.
 16. The method of claim 14, wherein the bacterial cells are from Neisseria or Francisella; or wherein the bacterial cells comprise recombinant vectors expressing one or a plurality of membrane-bound sugars and membrane-bound polypeptides or a combination of a membrane-bound sugar and a membrane-bound polypeptide from Neisseria or Francisella.
 17. The method of claim 14, wherein the aqueous solution comprises a marker or active agent.
 18. The method of claim 14, wherein the method further comprises filtering the vesicles through a size exclusion column or ion chromatography column.
 19. A method of creating a library of catanionic vesicles comprising: (a) adding a weight/weight molar ratio of cationic surfactant to anionic surfactant in aqueous solution of about 20:80 or about 80:20 to a population of bacterial cells in pelleted form; (b) stirring resulting mixture at room temperature for at least about 30 minutes; wherein the method does not comprise a step of purification prior to steps (a)-(b) or a step of removing lipopolysaccharides or lipooligosaccharides from the vesicles.
 20. The method of claim 19, wherein the cationic surfactant and anionic surfactant are SDBS and CTAT, respectively, at a molar weight ratio of about 70:30.
 21. The method of claim 19, wherein the bacterial cells are from Neisseria or Francisella; or wherein the bacterial cells comprise recombinant vectors expressing one or a plurality of membrane-bound sugars and membrane polypeptides or a combination of a membrane-bound sugar and a membrane-bound polypeptide from Neisseria or Francisella.
 22. The method of claim 19, wherein the aqueous solution comprises one or more of a dye, a fluorescent protein, or a chemiluminescent agent.
 23. The method of claim 14, wherein the method further comprises filtering the vesicles through a size exclusion column or ion chromatography column.
 24. A method of manufacturing an antibody against a bacterial antigen comprising injecting the composition of claim 1 into a subject and subsequently collecting its serum.
 25. The vaccine of claim 9, wherein the catanionic vesicles comprise a diameter of 300 nm or less and a carbohydrate percent from about 0.1% to about 15% by weight.
 26. The method of claim 14, wherein the bacterial cells are from Neisseria and Francisella.
 27. The method of claim 19, wherein the bacterial cells are from Neisseria or Francisella.
 28. The method of claim 16, wherein the membrane-bound polypeptide is from Neisseria.
 29. The method of claim 16, wherein the membrane-bound polypeptide is from Francisella.
 30. The method of claim 21, wherein the membrane-bound polypeptide is from Neisseria.
 31. The method of claim 21, wherein the membrane-bound polypeptide is from Francisella. 